Humanized antibodies that recognize beta amyloid peptide

ABSTRACT

The invention provides improved agents and methods for treatment of diseases associated with amyloid deposits of Aβ in the brain of a patient. Preferred agents include humanized antibodies.

RELATED APPLICATIONS

This application is a divisional of 10/388,214 filed, Mar. 12, 2003(pending), which claims the benefit of prior-filed provisional patentapplication U.S. Ser. No. 60/363,751 (filed Mar. 12, 2002) entitled“Humanized Antibodies That Recognize Beta-Amyloid Peptide”. The entirecontent of each of the above-referenced applications is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is a progressive disease resulting in seniledementia. See generally Selkoe, TINS 16:403 (1993); Hardy et al., WO92/13069; Selkoe, J. Neuropathol. Exp. Neurol. 53:438 (1994); Duff etal., Nature 373:476 (1995); Games et al., Nature 373:523 (1995). Broadlyspeaking, the disease falls into two categories: late onset, whichoccurs in old age (65+ years) and early onset, which develops wellbefore the senile period, i.e., between 35 and 60 years. In both typesof disease, the pathology is the same but the abnormalities tend to bemore severe and widespread in cases beginning at an earlier, age. Thedisease is characterized by at least two types of lesions in the brain,neurofibrillary tangles and senile plaques. Neurofibrillary tangles areintracellular deposits of microtubule associated tau protein consistingof two filaments twisted about each other in pairs. Senile plaques(i.e., amyloid plaques) are areas of disorganized neuropil up to 150 μmacross with extracellular amyloid deposits at the center which arevisible by microscopic analysis of sections of brain tissue. Theaccumulation of amyloid plaques within the brain is also associated withDown's syndrome and other cognitive disorders.

The principal constituent of the plaques is a peptide termed Aβ orβ-amyloid peptide. Aβ peptide is a 4-kDa internal fragment of 39-43amino acids of a larger transmembrane glycoprotein named protein termedamyloid precursor protein (APP). As a result of proteolytic processingof APP by different secretase enzymes, Aβ is primarily found in both ashort form, 40 amino acids in length, and a long form, ranging from42-43 amino acids in length. Part of the hydrophobic transmembranedomain of APP is found at the carboxy end of Aβ, and may account for theability of Aβ amyloid plaques in the brain eventually leads to neuronalcell death. The physical symptoms associated with this type of neuraldeterioration characterize Alzheimer's disease.

Several mutations within the APP protein have been correlated with thepresence of Alzheimer's disease. See, e.g., Goate et al., Nature349:704) (1991) (valine⁷¹⁷ to isoleucine); Chartier Harlan et al. Nature353:844 (1991)) (valine⁷¹⁷ to glycine); Murrell et al., Science 254:97(1991) (valine⁷¹⁷ to phenylalanine); Mullan et al., Nature Genet. 1:345(1992) (a double mutation changing lysine⁵⁹⁵-methionine⁵⁹⁶ toasparagine⁵⁹⁵-leucine⁵⁹⁶). Such mutations are thought to causeAlzheimer's disease by increased or altered processing of APP to Aβ,particularly processing of APP to increased amounts of the long form ofAβ (i.e., Aβ1-42 and Aβ1-43). Mutations in other genes, such as thepresenilin genes, PS1 and PS2, are thought indirectly to affectprocessing of APP to generate increased amounts of long form Aβ (seeHardy, TINS 20: 154 (1997)).

Mouse models have been used successfully to determine the significanceof amyloid plaques in Alzheimer's (Games et al., supra, Johnson-Wood etal., Proc. Natl. Acad. Sci. USA 94:1550 (1997)). In particular, whenPDAPP transgenic mice, (which express a mutant form of human APP anddevelop Alzheimer's disease at a young age), are injected with the longform of Aβ, they display both a decrease in the progression ofAlzheimer's and an increase in antibody titers to the Aβ peptide (Schenket al., Nature 400, 173 (1999)). The observations discussed aboveindicate that Aβ, particularly in its long form, is a causative elementin Alzheimer's disease.

Accordingly, there exists the need for new therapies and reagents forthe treatment of Alzheimer's disease, in particular, therapies andreagents capable of effecting a therapeutic benefit at physiologic(e.g., non-toxic) doses.

SUMMARY OF THE INVENTION

The present invention features new immunological reagents, inparticular, therapeutic antibody reagents for the prevention andtreatment of amyloidogenic disease (e.g., Alzheimer's disease). Theinvention is based, at least in part, on the identification andcharacterization of a monoclonal antibody that specifically binds to Aβpeptide and is effective at reducing plaque burden and/or reducing theneuritic dystrophy associated with amyloidogenic disorders. Structuraland functional analysis of this antibody leads to the design of varioushumanized antibodies for prophylactic and/or therapeutic use. Inparticular, the invention features humanization of the variable regionsof this antibody and, accordingly, provides for humanized immunoglobulinor antibody chains, intact humanized immunoglobulins or antibodies, andfunctional immunoglobulin or antibody fragments, in particular, antigenbinding fragments, of the featured antibody.

Polypeptides comprising the complementarity determining regions of thefeatured monoclonal antibody are also disclosed, as are polynucleotidereagents, vectors and host cells suitable for encoding saidpolypeptides.

Methods of treatment of amyloidogenic diseases or disorders (e.g.,Alzheimer's disease) are disclosed, as are pharmaceutical compositionsand kits for use in such applications.

Also featured are methods of identifying residues within the featuredmonoclonal antibodies which are important for proper immunologicfunction and for identifying residues which are amenable to substitutionin the design of humanized antibodies having improved binding affinitiesand/or reduced immunogenicity, when used as therapeutic reagents.

Also featured are antibodies (e.g., humanized antibodies) having alteredeffector functions, and therapeutic uses thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B depicts an alignment of the amino acid sequences of the lightchain of mouse 12B4 (mature peptide, SEQ ID NO:2), humanized 12B4(mature peptide, SEQ ID NO:6), Kabat ID 005036 (mature peptide, SEQ IDNO:32) and germline A19 (X63397, mature peptide, SEQ ID NO:30)antibodies. CDR regions are stippled and overlined. The singlebackmutation of a human—÷ mouse residue is indicated by the asterisk.The importance of the shaded residues is shown in the legend. Numberedfrom the first methionine, not Kabat numbering.

FIG. 2A-B depicts an alignment of the amino acid sequences of the heavychain of mouse 12B4 (mature peptide, SEQ ID NO:4), humanized 12B4(version 1) (mature peptide, SEQ ID NO:8), Kabat ID 000333 (maturepeptide, SEQ ID NO:34), and germline VH4-39 and VH4-61 antibodies(mature peptides, SEQ ID NOs: 38 and 36, respectively). Annotation isthe same as for FIG. 1. Numbered from the first methionine, not Kabatnumbering.

FIG. 3A-D depicts the nucleotide and amino acid sequence for humanized12B4VLv1 compared with chimeric 12B4VL (identical variable regionsequences as murine 12B4VL, SEQ ID NOs: 1 and 2, respectively); germlineA19 sequences (SEQ ID NOs: 29 and 30, respectively); and Kabid ID 005036(SEQ ID NOs: 31 and 32, respectively).

FIG. 4A-D depicts the nucleotide and amino acid sequence for humanized12B4VHv1 compared with chimeric 12B4VH (identical variable regionsequences as murine 12B4VH, SEQ ID NOs: 3 and 4, respectively); Kabat ID000333 (SEQ ID NOs: 33 and 34, respectively); and germline VH4-61 (SEQID NOs: 35 and 36, respectively).

FIG. 5 graphically depicts the ELISA results from two independentexperiments measuring the binding of chimeric 12B4, 3D6, and chimeric3D6 to Aβ (panels A and B, respectively).

FIG. 6 graphically depicts competitive ELISA binding confirmingfunctional activity of 12B4 and chimeric 12B4 as compared to 3D6,chimeric 3D6, and 10D5. Chimeric 12B4 (open triangles) competes withequal potency with its non biotinylated murine counterpart (openinverted triangles) for binding of biotinylated murine 12B4 to Aβ1-42peptide.

FIG. 7 graphically depicts an ex vivo phagocytosis assay testing theability of chimeric 12B4, 3D6, and human IgG1 to mediate the uptake ofAβ by microglial cells on PDAPP brain sections.

FIG. 8 graphically depicts the results from two independent ex vivophagocytosis assays (panels A and B, respectively) testing the abilityof chimeric 12B4, humanized 3D6, and human IgG1 to mediate the uptake ofAβ by microglial cells on AD brain sections.

FIG. 9 is a schematic representation of the PCR-mediated assembly ofhumanized 12B4, version 1. FIG. 9A depicts the assembly of the VLregions. FIG. 9B depicts the assembly of the VH regions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention features new immunological reagents and methodsfor preventing or treating Alzheimer's disease or other amyloidogenicdiseases. The invention is based, at least in part, on thecharacterization of a monoclonal immunoglobulin, 12B4, effective atbinding beta amyloid protein (Aβ) (e.g., binding soluble and/oraggregated Aβ), mediating phagocytosis (e.g., of aggregated Aβ),reducing plaque burden and/or reducing neuritic dystrophy (e.g., in apatient). The invention is further based on the determination andstructural characterization of the primary and secondary structure ofthe variable light and heavy chains of the 12B4 immunoglobulin and theidentification of residues important for activity and immunogenicity.

Immunoglobulins are featured which include a variable light and/orvariable heavy chain of the 12B4 monoclonal immunoglobulin describedherein. Preferred immunoglobulins, e.g., therapeutic immunoglobulins,are featured which include a humanized variable light and/or humanizedvariable heavy chain. Preferred variable light and/or variable heavychains include a complementarity determining region (CDR) from the 12B4immunoglobulin (e.g., donor immunoglobulin) and variable frameworkregions substantially from a human acceptor immunoglobulin. The phrase“substantially from a human acceptor immunoglobulin” means that themajority or key framework residues are from the human acceptor sequence,allowing however, for substitution of residues at certain positions withresidues selected to improve activity of the humanized immunoglobulin(e.g., alter activity such that it more closely mimics the activity ofthe donor immunoglobulin) or selected to decrease the immunogenicity ofthe humanized immunoglobulin.

In one embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 12B4 variable region complementaritydetermining regions (CDRs) (i.e., includes one, two or three CDRs fromthe light chain variable region sequence set forth as SEQ ID NO:2 orincludes one, two or three CDRs from the heavy chain variable regionsequence set forth as SEQ ID NO:4), and includes a variable frameworkregion substantially from a human acceptor immunoglobulin light or heavychain sequence, provided that at least one residue of the frameworkresidue is backmutated to a corresponding murine residue, wherein saidbackmutation does not substantially affect the ability of the chain todirect Aβ binding.

In another embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 12B4 variable region complementaritydetermining regions (CDRs) (e.g., includes one, two or three CDRs fromthe light chain variable region sequence set forth as SEQ ID NO:2 and/orincludes one, two or three CDRs from the heavy chain variable regionsequence set forth as SEQ ID NO:4), and includes a variable frameworkregion substantially from a human acceptor immunoglobulin light or heavychain sequence, provided that at least one framework residue issubstituted with the corresponding amino acid residue from the mouse12B4 light or heavy chain variable region sequence, where the frameworkresidue is selected from the group consisting of (a) a residue thatnon-covalently binds antigen directly; (b) a residue adjacent to a CDR;(c) a CDR-interacting residue (e.g., identified by modeling the light orheavy chain on the solved structure of a homologous known immunoglobulinchain); and (d) a residue participating in the VL-VH interface.

In another embodiment, the invention features a humanized immunoglobulinlight or heavy chain that includes 12B4 variable region CDRs andvariable framework regions from a human acceptor immunoglobulin light orheavy chain sequence, provided that at least one framework residue issubstituted with the corresponding amino acid residue from the mouse12B4 light or heavy chain variable region sequence, where the frameworkresidue is a residue capable of affecting light chain variable regionconformation or function as identified by analysis of athree-dimensional model of the variable region, for example a residuecapable of interacting with antigen, a residue proximal to the antigenbinding site, a residue capable of interacting with a CDR, a residueadjacent to a CDR, a residue within 6 Å of a CDR residue, a canonicalresidue, a vernier zone residue, an interchain packing residue, anunusual residue, or a glycoslyation site residue on the surface of thestructural model.

In another embodiment, the invention features, in addition to thesubstitutions described above, a substitution of at least one rare humanframework residue. For example, a rare residue can be substituted withan amino acid residue which is common for human variable chain sequencesat that position. Alternatively, a rare residue can be substituted witha corresponding amino acid residue from a homologous germline variablechain sequence.

In another embodiment, the invention features a humanized immunoglobulinthat includes a light chain and a heavy chain, as described above, or anantigen-binding fragment of said immunoglobulin. In an exemplaryembodiment, the humanized immunoglobulin binds (e.g., specificallybinds) to beta amyloid peptide (Aβ) with a binding affinity of at least10⁷ M⁻¹, 10⁸M⁻¹, or 10⁹ M⁻¹. In another embodiment, the immunoglobulinor antigen binding fragment includes a heavy chain having isotype γ1. Inanother embodiment, the immunoglobulin or antigen binding fragment binds(e.g., specifically binds) to both soluble beta amyloid peptide (Aβ) andaggregated A. In another embodiment, the immunoglobulin or antigenbinding fragment mediates phagocytosis (e.g., induces phagocytosis) ofbeta amyloid peptide (Aβ). In yet another embodiment, the immunoglobulinor antigen binding fragment crosses the blood-brain barrier in asubject. In yet another embodiment, the immunoglobulin or antigenbinding fragment reduces both beta amyloid peptide (Aβ) burden andneuritic dystrophy in a subject.

In another embodiment, the invention features chimeric immunoglobulinsthat include 12B4 variable regions (e.g., the variable region sequencesset forth as SEQ ID NO:2 or SEQ ID NO:4). In yet another embodiment, theimmunoglobulin, or antigen-binding fragment thereof, further includesconstant regions from IgG1.

The immunoglobulins described herein are particularly suited for use intherapeutic methods aimed at preventing or treating amyloidogenicdiseases. In one embodiment, the invention features a method ofpreventing or treating an amyloidogenic disease (e.g., Alzheimer'sdisease) that involves administering to the patient an effective dosageof a humanized immunoglobulin as described herein. In anotherembodiment, the invention features pharmaceutical compositions thatinclude a humanized immunoglobulin as described herein and apharmaceutical carrier. Also featured are isolated nucleic acidmolecules, vectors and host cells for producing the immunoglobulins orimmunoglobulin fragments or chains described herein, as well as methodsfor producing said immunoglobulins, immunoglobulin fragments orimmunoglobulin chains

The present invention further features a method for identifying 12B4residues amenable to substitution when producing a humanized 12B4immunoglobulin, respectively. For example, a method for identifyingvariable framework region residues amenable to substitution involvesmodeling the three-dimensional structure of a 12B4 variable region on asolved homologous immunoglobulin structure and analyzing said model forresidues capable of affecting 12B4 immunoglobulin variable regionconformation or function, such that residues amenable to substitutionare identified. The invention further features use of the variableregion sequence set forth as SEQ ID NO:2 or SEQ ID NO:4, or any portionthereof, in producing a three-dimensional image of a 12B4immunoglobulin, 12B4 immunoglobulin chain, or domain thereof.

The present invention further features immunoglobulins having alteredeffector function, such as the ability to bind effector molecules, forexample, complement or a receptor on an effector cell. In particular,the immunoglobulin of the invention has an altered constant region,e.g., Fc region, wherein at least one amino acid residue in the Fcregion has been replaced with a different residue or side chain. In oneembodiment, the modified immunoglobulin is of the IgG class, comprisesat least one amino acid residue replacement in the Fc region such thatthe immunoglobulin has an altered effector function, e.g., as comparedwith an unmodified immunoglobulin. In particular embodiments, theimmunoglobulin of the invention has an altered effector function suchthat it is less immunogenic (e.g., does not provoke undesired effectorcell activity, lysis, or complement binding), has improved amyloidclearance properties, and/or has a desirable half-life.

Prior to describing the invention, it may be helpful to an understandingthereof to set forth definitions of certain terms to be usedhereinafter.

The term “immunoglobulin” or “antibody” (used interchangeably herein)refers to a protein having a basic four-polypeptide chain structureconsisting of two heavy and two light chains, said chains beingstabilized, for example, by interchain disulfide bonds, which has theability to specifically bind antigen. The term “single-chainimmunoglobulin” or “single-chain antibody” (used interchangeably herein)refers to a protein having a two-polypeptide chain structure consistingof a heavy and a light chain, said chains being stabilized, for example,by interchain peptide linkers, which has the ability to specificallybind antigen. The term “domain” refers to a globular region of a heavyor light chain polypeptide comprising peptide loops (e.g., comprising 3to 4 peptide loops) stabilized, for example, by β-pleated sheet and/orintrachain disulfide bond. Domains are further referred to herein as“constant” or “variable”, based on the relative lack of sequencevariation within the domains of various class members in the case of a“constant” domain, or the significant variation within the domains ofvarious class members in the case of a “variable” domain. Antibody orpolypeptide “domains” are often referred to interchangeably in the artas antibody or polypeptide “regions”. The “constant” domains of anantibody light chain are referred to interchangeably as “light chainconstant regions”, “light chain constant domains”, “CL” regions or “CL”domains. The “constant” domains of an antibody heavy chain are referredto interchangeably as “heavy chain constant regions”, “heavy chainconstant domains”, “CH” regions or “CH” domains). The “variable” domainsof an antibody light chain are referred to interchangeably as “lightchain variable regions”, “light chain variable domains”, “VL” regions or“VL” domains). The “variable” domains of an antibody heavy chain arereferred to interchangeably as “heavy chain constant regions”, “heavychain constant domains”, “CH” regions or “CH” domains).

The term “region” can also refer to a part or portion of an antibodychain or antibody chain domain (e.g., a part or portion of a heavy orlight chain or a part or portion of a constant or variable domain, asdefined herein), as well as more discrete parts or portions of saidchains or domains. For example, light and heavy chains or light andheavy chain variable domains include “complementarity determiningregions” or “CDRs” interspersed among “framework regions” or “FRs”, asdefined herein.

Immunoglobulins or antibodies can exist in monomeric or polymeric form,for example, IgM antibodies which exist in pentameric form and/or IgAantibodies which exist in monomeric, dimeric or multimeric form. Theterm “fragment” refers to a part or portion of an antibody or antibodychain comprising fewer amino acid residues than an intact or completeantibody or antibody chain. Fragments can be obtained via chemical orenzymatic treatment of an intact or complete antibody or antibody chain.Fragments can also be obtained by recombinant means. Exemplary fragmentsinclude Fab, Fab′, F(ab′)2, Fabc and/or Fv fragments. The term“antigen-binding fragment” refers to a polypeptide fragment of animmunoglobulin or antibody that binds antigen or competes with intactantibody (i.e., with the intact antibody from which they were derived)for antigen binding (i.e., specific binding). The term “conformation”refers to the tertiary structure of a protein or polypeptide (e.g., anantibody, antibody chain, domain or region thereof). For example, thephrase “light (or heavy) chain conformation” refers to the tertiarystructure of a light (or heavy) chain variable region, and the phrase“antibody conformation” or “antibody fragment conformation” refers tothe tertiary structure of an antibody or fragment thereof.

“Specific binding” of an antibody mean that the antibody exhibitsappreciable affinity for antigen or a preferred epitope and, preferably,does not exhibit significant crossreactivity. “Appreciable” or preferredbinding include binding with an affinity of at least 10⁶, 10⁷, 10⁸, 10⁹M⁻¹, or 10¹⁰ M⁻¹. Affinities greater than 10⁷M⁻¹, preferably greaterthan 10⁸ M⁻¹ are more preferred. Values intermediate of those set forthherein are also intended to be within the scope of the present inventionand a preferred binding affinity can be indicated as a range ofaffinities, for example, 10⁶ to 10¹⁰ M⁻¹, preferably 10⁷ to 10¹⁰ M⁻¹,more preferably 10⁸ to 10¹⁰ M⁻¹. An antibody that “does not exhibitsignificant crossreactivity” is one that will not appreciably bind to anundesirable entity (e.g., an undesirable proteinaceous entity). Forexample, an antibody that specifically binds to Aβ will appreciably bindAβ but will not significantly react with non-Aβ proteins or peptides(e.g., non-Aβ proteins or peptides included in plaques). An antibodyspecific for a preferred epitope will, for example, not significantlycrossreact with remote epitopes on the same protein or peptide. Specificbinding can be determined according to any art-recognized means fordetermining such binding. Preferably, specific binding is determinedaccording to Scatchard analysis and/or competitive binding assays.

Binding fragments are produced by recombinant DNA techniques, or byenzymatic or chemical cleavage of intact immunoglobulins. Bindingfragments include Fab, Fab′, F(ab′)₂, Fabc, Fv, single chains, andsingle-chain antibodies. Other than “bispecific” or “bifunctional”immunoglobulins or antibodies, an immunoglobulin or antibody isunderstood to have each of its binding sites identical. A “bispecific”or “bifunctional antibody” is an artificial hybrid antibody having twodifferent heavy/light chain pairs and two different binding sites.Bispecific antibodies can be produced by a variety of methods includingfusion of hybridomas or linking of Fab′ fragments. See, e.g.,Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelnyet al., J. Immunol. 148, 1547-1553 (1992).

The term “humanized immunoglobulin” or “humanized antibody” refers to animmunoglobulin or antibody that includes at least one humanizedimmunoglobulin or antibody chain (i.e., at least one humanized light orheavy chain). The term “humanized immunoglobulin chain” or “humanizedantibody chain” (i.e., a “humanized immunoglobulin light chain” or“humanized immunoglobulin heavy chain”) refers to an immunoglobulin orantibody chain (i.e., a light or heavy chain, respectively) having avariable region that includes a variable framework region substantiallyfrom a human immunoglobulin or antibody and complementarity determiningregions (CDRs) (e.g., at least one CDR, preferably two CDRs, morepreferably three CDRs) substantially from a non-human immunoglobulin orantibody, and further includes constant regions (e.g., at least oneconstant region or portion thereof, in the case of a light chain, andpreferably three constant regions in the case of a heavy chain). Theterm “humanized variable region” (e.g., “humanized light chain variableregion” or “humanized heavy chain variable region”) refers to a variableregion that includes a variable framework region substantially from ahuman immunoglobulin or antibody and complementarity determining regions(CDRs) substantially from a non-human immunoglobulin or antibody.

The phrase “substantially from a human immunoglobulin or antibody” or“substantially human” means that, when aligned to a human immunoglobulinor antibody amino sequence for comparison purposes, the region shares atleast 80-90%, preferably at least 90-95%, more preferably at least95-99% identity (i.e., local sequence identity) with the human frameworkor constant region sequence, allowing, for example, for conservativesubstitutions, consensus sequence substitutions, germline substitutions,backmutations, and the like. The introduction of conservativesubstitutions, consensus sequence substitutions, germline substitutions,backmutations, and the like, is often referred to as “optimization” of ahumanized antibody or chain. The phrase “substantially from a non-humanimmunoglobulin or antibody” or “substantially non-human” means having animmunoglobulin or antibody sequence at least 80-95%, preferably at least90-95%, more preferably, 96%, 97%, 98%, or 99% identical to that of anon-human organism, e.g., a non-human mammal.

Accordingly, all regions or residues of a humanized immunoglobulin orantibody, or of a humanized immunoglobulin or antibody chain, exceptpossibly the CDRs, are substantially identical to the correspondingregions or residues of one or more native human immunoglobulinsequences. The term “corresponding region” or “corresponding residue”refers to a region or residue on a second amino acid or nucleotidesequence which occupies the same (i.e., equivalent) position as a regionor residue on a first amino acid or nucleotide sequence, when the firstand second sequences are optimally aligned for comparison purposes.

The term “significant identity” means that two polypeptide sequences,when optimally aligned, such as by the programs GAP or BESTFIT usingdefault gap weights, share at least 50-60% sequence identity, preferablyat least 60-70% sequence identity, more preferably at least 70-80%sequence identity, more preferably at least 80-90% identity, even morepreferably at least 90-95% identity, and even more preferably at least95% sequence identity or more (e.g., 99% sequence identity or more). Theterm “substantial identity” means that two polypeptide sequences, whenoptimally aligned, such as by the programs GAP or BESTFIT using defaultgap weights, share at least 80-90% sequence identity, preferably atleast 90-95% sequence identity, and more preferably at least 95%sequence identity or more (e.g., 99% sequence identity or more). Forsequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., Current Protocols in Molecular Biology). One example ofalgorithm that is suitable for determining percent sequence identity andsequence similarity is the BLAST algorithm, which is described inAltschul et al., J. Mol. Biol. 215:403 (1990). Software for performingBLAST analyses is publicly available through the National Center forBiotechnology Information (publicly accessible through the NationalInstitutes of Health NCBI internet server). Typically, default programparameters can be used to perform the sequence comparison, althoughcustomized parameters can also be used. For amino acid sequences, theBLASTP program uses as defaults a wordlength (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff,Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

Preferably, residue positions which are not identical differ byconservative amino acid substitutions. For purposes of classifying aminoacids substitutions as conservative or nonconservative, amino acids aregrouped as follows: Group I (hydrophobic sidechains): leu, met, ala,val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser,thr; Group III (acidic side chains): asp, glu; Group IV (basic sidechains): asn, gln, his, lys, arg; Group V (residues influencing chainorientation): gly, pro; and Group VI (aromatic side chains): trp, tyr,phe. Conservative substitutions involve substitutions between aminoacids in the same class. Non-conservative substitutions constituteexchanging a member of one of these classes for a member of another.

Preferably, humanized immunoglobulins or antibodies bind antigen with anaffinity that is within a factor of three, four, or five of that of thecorresponding non-humanized antibody. For example, if the nonhumanizedantibody has a binding affinity of 10⁹ M⁻¹, humanized antibodies willhave a binding affinity of at least 3×10⁹ M⁻¹, 4×10⁹ M⁻¹ or 10⁹ M⁻¹.When describing the binding properties of an immunoglobulin or antibodychain, the chain can be described based on its ability to “directantigen (e.g., Aβ) binding”. A chain is said to “direct antigen binding”when it confers upon an intact immunoglobulin or antibody (or antigenbinding fragment thereof) a specific binding property or bindingaffinity. A mutation (e.g., a backmutation) is said to substantiallyaffect the ability of a heavy or light chain to direct antigen bindingif it affects (e.g., decreases) the binding affinity of an intactimmunoglobulin or antibody (or antigen binding fragment thereof)comprising said chain by at least an order of magnitude compared to thatof the antibody (or antigen binding fragment thereof) comprising anequivalent chain lacking said mutation. A mutation “does notsubstantially affect (e.g., decrease) the ability of a chain to directantigen binding” if it affects (e.g., decreases) the binding affinity ofan intact immunoglobulin or antibody (or antigen binding fragmentthereof) comprising said chain by only a factor of two, three, or fourof that of the antibody (or antigen binding fragment thereof) comprisingan equivalent chain lacking said mutation.

The term “chimeric immunoglobulin” or antibody refers to animmunoglobulin or antibody whose variable regions derive from a firstspecies and whose constant regions derive from a second species.Chimeric immunoglobulins or antibodies can be constructed, for exampleby genetic engineering, from immunoglobulin gene segments belonging todifferent species. The terms “humanized immunoglobulin” or “humanizedantibody” are not intended to encompass chimeric immunoglobulins orantibodies, as defined infra. Although humanized immunoglobulins orantibodies are chimeric in their construction (i.e., comprise regionsfrom more than one species of protein), they include additional features(i.e., variable regions comprising donor CDR residues and acceptorframework residues) not found in chimeric immunoglobulins or antibodies,as defined herein.

An “antigen” is an entity (e.g., a proteinaceous entity or peptide) towhich an antibody specifically binds.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which an immunoglobulin or antibody (or antigen bindingfragment thereof) specifically binds. Epitopes can be formed both fromcontiguous amino acids or noncontiguous amino acids juxtaposed bytertiary folding of a protein. Epitopes formed from contiguous aminoacids are typically retained on exposure to denaturing solvents, whereasepitopes formed by tertiary folding are typically lost on treatment withdenaturing solvents. An epitope typically includes at least 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatialconformation. Methods of determining spatial conformation of epitopesinclude, for example, x-ray crystallography and 2-dimensional nuclearmagnetic resonance. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

Antibodies that recognize the same epitope can be identified in a simpleimmunoassay showing the ability of one antibody to block the binding ofanother antibody to a target antigen, i.e., a competitive binding assay.Competitive binding is determined in an assay in which theimmunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as Aβ. Numerous types of competitivebinding assays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidinEIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress (1988)); solid phase direct label RIA using 1-125 label (see Morelet al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidinEIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, suchan assay involves the use of purified antigen bound to a solid surfaceor cells bearing either of these, an unlabeled test immunoglobulin and alabeled reference immunoglobulin. Competitive inhibition is measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test immunoglobulin. Usually the test immunoglobulinis present in excess. Usually, when a competing antibody is present inexcess, it will inhibit specific binding of a reference antibody to acommon antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% ormore.

An epitope is also recognized by immunologic cells, for example, B cellsand/or T cells. Cellular recognition of an epitope can be determined byin vitro assays that measure antigen-dependent proliferation, asdetermined by ³H-thymidine incorporation, by cytokine secretion, byantibody secretion, or by antigen-dependent killing (cytotoxic Tlymphocyte assay).

Exemplary epitopes or antigenic determinants can be found within thehuman amyloid precursor protein (APP), but are preferably found withinthe Aβ peptide of APP. Multiple isoforms of APP exist, for exampleAPP⁶⁹⁵ APP⁷⁵¹ and APP⁷⁷⁰. Amino acids within APP are assigned numbersaccording to the sequence of the APP⁷⁷⁰ isoform (see e.g., GenBankAccession No. P05067). Aβ (also referred to herein as beta amyloidpeptide and A-beta) peptide is an approximately 4-kDa internal fragmentof 39-43 amino acids of APP (Aβ39, Aβ40, Aβ41, Aβ42 and Aβ43). Aβ40, forexample, consists of residues 672-711 of APP and Aβ42 consists ofresidues 673-713 of APP. As a result of proteolytic processing of APP bydifferent secretase enzymes iv vivo or in situ, Aβ is found in both a“short form”, 40 amino acids in length, and a “long form”, ranging from42-43 amino acids in length. Preferred epitopes or antigenicdeterminants, as described herein, are located within the N-terminus ofthe Aβ peptide and include residues within amino acids 1-10 of Aβ,preferably from residues 1-3, 1-4, 1-5, 1-6, 1-7 or 3-7 of Aβ42.Additional referred epitopes or antigenic determinants include residues2-4, 5, 6, 7 or 8 of Aβ, residues 3-5, 6, 7, 8 or 9 of Aβ, or residues4-7, 8, 9 or 10 of Aβ42. When an antibody is said to bind to an epitopewithin specified residues, such as Aβ 3-7, what is meant is that theantibody specifically binds to a polypeptide containing the specifiedresidues (i.e., Aβ 3-7 in this an example). Such an antibody does notnecessarily contact every residue within Aβ 3-7. Nor does every singleamino acid substitution or deletion with in Aβ 3-7 necessarilysignificantly affect binding affinity.

The term “amyloidogenic disease” includes any disease associated with(or caused by) the formation or deposition of insoluble amyloid fibrils.Exemplary amyloidogenic diseases include, but are not limited tosystemic amyloidosis, Alzheimer's disease, mature onset diabetes,Parkinson's disease, Huntington's disease, fronto-temporal dementia, andthe prion-related transmissible spongiform encephalopathies (kuru andCreutzfeldt-Jacob disease in humans and scrapie and BSE in sheep andcattle, respectively). Different amyloidogenic diseases are defined orcharacterized by the nature of the polypeptide component of the fibrilsdeposited. For example, in subjects or patients having Alzheimer'sdisease, β-amyloid protein (e.g., wild-type, variant, or truncatedβ-amyloid protein) is the characterizing polypeptide component of theamyloid deposit. Accordingly, Alzheimer's disease is an example of a“disease characterized by deposits of Aβ” or a “disease associated withdeposits of Aβ”, e.g., in the brain of a subject or patient. The terms“β-amyloid protein”, “β-amyloid peptide”, “β-amyloid”, “Aβ” and “Aβpeptide” are used interchangeably herein.

An “immunogenic agent” or “immunogen” is capable of inducing animmunological response against itself on administration to a mammal,optionally in conjunction with an adjuvant.

The term “treatment” as used herein, is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease or the predisposition toward disease.

The term “effective dose” or “effective dosage” is defined as an amountsufficient to achieve or at least partially achieve the desired effect.The term “therapeutically effective dose” is defined as an amountsufficient to cure or at least partially arrest the disease and itscomplications in a patient already suffering from the disease. Amountseffective for this use will depend upon the severity of the infectionand the general state of the patient's own immune system.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

“Soluble” or “dissociated” Aβ refers to non-aggregating or disaggregatedAβ polypeptide, including monomeric soluble as well as oligomericsoluble Aβ polypeptide (e.g., soluble Aβ dimers, trimers, and the like).“Insoluble” Aβ refers to aggregating Aβ polypeptide, for example, Aβheld together by noncovalent bonds. Aβ (e.g., Aβ42) is believed toaggregate, at least in part, due to the presence of hydrophobic residuesat the C-terminus of the peptide (part of the transmembrane domain ofAPP). Soluble Aβ can be found in vivo in biological fluids such ascerebrospinal fluid and/or serum. Alternatively, soluble Aβ can beprepared by dissolving lyophilized peptide in neat DMSO with sonication.The resulting solution is centrifuged (e.g., at 14,000×g, 4° C., 10minutes) to remove any insoluble particulates.

The term “effector function” refers to an activity that resides in theFc region of an antibody (e.g., an IgG antibody) and includes, forexample, the ability of the antibody to bind effector molecules such ascomplement and/or Fc receptors, which can control several immunefunctions of the antibody such as effector cell activity, lysis,complement-mediated activity, antibody clearance, and antibodyhalf-life.

The term “effector molecule” refers to a molecule that is capable ofbinding to the Fc region of an antibody (e.g., an IgG antibody)including, but not limited to, a complement protein or a Fc receptor.

The term “effector cell” refers to a cell capable of binding to the Fcportion of an antibody (e.g., an IgG antibody) typically via an Fcreceptor expressed on the surface of the effector cell including, butnot limited to, lymphocytes, e.g., antigen presenting cells and T cells.

The term “Fc region” refers to a C-terminal region of an IgG antibody,in particular, the C-terminal region of the heavy chain(s) of said IgGantibody. Although the boundaries of the Fc region of an IgG heavy chaincan vary slightly, a Fc region is typically defined as spanning fromabout amino acid residue Cys226 to the carboxyl-terminus of an IGg heavychain(s).

The term “Kabat numbering” unless otherwise stated, is defined as thenumbering of the residues in, e.g., an IgG heavy chain antibody usingthe EU index as in Kabat et al. (Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991)), expressly incorporated herein by reference.

The term “Fc receptor” or “FcR” refers to a receptor that binds to theFc region of an antibody. Typical Fc receptors which bind to an Fcregion of an antibody (e.g., an IgG antibody) include, but are notlimited to, receptors of the FcγRI, FcγRII, and FcγRIII subclasses,including allelic variants and alternatively spliced forms of thesereceptors. Fc receptors are reviewed in Ravetch and Kinet, Arum. Rev.Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); andde Haas et al., J. Lab. Clin. Med. 126:330-41 (1995).

I. Immunological and Therapeutic Reagents

Immunological and therapeutic reagents of the invention comprise orconsist of immunogens or antibodies, or functional or antigen bindingfragments thereof, as defined herein. The basic antibody structural unitis known to comprise a tetramer of subunits. Each tetramer is composedof two identical pairs of polypeptide chains, each pair having one“light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function.

Light chains are classified as either kappa or lambda and are about 230residues in length. Heavy chains are classified as gamma (γ), mu (μ),alpha (α), delta (δ), or epsilon (ε), are about 450-600 residues inlength, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE,respectively. Both heavy and light chains are folded into domains. Theterm “domain” refers to a globular region of a protein, for example, animmunoglobulin or antibody. Immunoglobulin or antibody domains include,for example, 3 or four peptide loops stabilized by 3-pleated sheet andan interchain disulfide bond. Intact light chains have, for example, twodomains (V_(L) and C_(L)) and intact heavy chains have, for example,four or five domains (V_(H), C_(H)1, C_(H)2, and C_(H)3).

Within light and heavy chains, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 10 more amino acids. (Seegenerally, Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press,N.Y. (1989), Ch. 7, incorporated by reference in its entirety for allpurposes).

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, an intact antibody has two binding sites. Except inbifunctional or bispecific antibodies, the two binding sites are thesame. The chains all exhibit the same general structure of relativelyconserved framework regions (FR) joined by three hypervariable regions,also called complementarity determining regions or CDRs.Naturally-occurring chains or recombinantly produced chains can beexpressed with a leader sequence which is removed during cellularprocessing to produce a mature chain. Mature chains can also berecombinantly produced having a non-naturally occurring leader sequence,for example, to enhance secretion or alter the processing of aparticular chain of interest.

The CDRs of the two mature chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. “FR4” also is referredto in the art as the D/J region of the variable heavy chain and the Jregion of the variable light chain. The assignment of amino acids toeach domain is in accordance with the definitions of Kabat, Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md., 1987 and 1991). An alternative structural definition hasbeen proposed by Chothia et al., J. Mol. Biol. 196:901 (1987); Nature342:878 (1989); and J. Mol. Biol. 186:651 (1989) (hereinaftercollectively referred to as “Chothia et al.” and incorporated byreference in their entirety for all purposes).

A. Aβ Antibodies

Therapeutic agents of the invention include antibodies that specificallybind to Aβ or to other components of the amyloid plaque. Preferredantibodies are monoclonal antibodies. Some such antibodies bindspecifically to the aggregated form of Aβ without binding to the solubleform. Some bind specifically to the soluble form without binding to theaggregated form. Some bind to both aggregated and soluble forms.Antibodies used in therapeutic methods preferably have an intactconstant region or at least sufficient of the constant region tointeract with an Fc receptor. Preferred antibodies are those efficaciousat stimulating Fc-mediated phagocytosis of Aβ in plaques. Human isotypeIgG1 is preferred because of it having highest affinity of humanisotypes for the FcRI receptor on phagocytic cells (e.g., on brainresident macrophages or microglial cells). Human IgG1 is the equivalentof murine IgG2a, the latter thus suitable for testing in vivo efficacyin animal (e.g., mouse) models of Alzheimer's. Bispecific Fab fragmentscan also be used, in which one arm of the antibody has specificity forAβ, and the other for an Fc receptor. Preferred antibodies bind to Aβwith a binding affinity greater than (or equal to) about 10⁶, 10⁷, 10⁸,10⁹, or 10¹⁰ M⁻¹ (including affinities intermediate of these values).

Monoclonal antibodies bind to a specific epitope within Aβ that can be aconformational or nonconformational epitope. Prophylactic andtherapeutic efficacy of antibodies can be tested using the transgenicanimal model procedures described in the Examples. Preferred monoclonalantibodies bind to an epitope within residues 1-10 of Aβ (with the firstN terminal residue of natural Aβ designated 1), more preferably to anepitope within residues 3-7 of Aβ. In some methods, multiple monoclonalantibodies having binding specificities to different epitopes are used,for example, an antibody specific for an epitope within residues 3-7 ofAβ can be co-administered with an antibody specific for an epitopeoutside of residues 3-7 of Aβ. Such antibodies can be administeredsequentially or simultaneously. Antibodies to amyloid components otherthan Aβ can also be used (e.g., administered or co-administered).

Epitope specificity of an antibody can be determined, for example, byforming a phage display library in which different members displaydifferent subsequences of Aβ. The phage display library is then selectedfor members specifically binding to an antibody under test. A family ofsequences is isolated. Typically, such a family contains a common coresequence, and varying lengths of flanking sequences in differentmembers. The shortest core sequence showing specific binding to theantibody defines the epitope bound by the antibody. Antibodies can alsobe tested for epitope specificity in a competition assay with anantibody whose epitope specificity has already been determined. Forexample, antibodies that compete with the 12B4 antibody for binding toAβ bind to the same or similar epitope as 12B4, i.e., within residues Aβ3-7. Screening antibodies for epitope specificity is a useful predictorof therapeutic efficacy. For example, an antibody determined to bind toan epitope within residues 1-7 of Aβ is likely to be effective inpreventing and treating Alzheimer's disease according to themethodologies of the present invention.

Antibodies that specifically bind to a preferred segment of Aβ withoutbinding to other regions of Aβ have a number of advantages relative tomonoclonal antibodies binding to other regions or polyclonal sera tointact Aβ. First, for equal mass dosages, dosages of antibodies thatspecifically bind to preferred segments contain a higher molar dosage ofantibodies effective in clearing amyloid plaques. Second, antibodiesspecifically binding to preferred segments can induce a clearingresponse against amyloid deposits without inducing a clearing responseagainst intact APP polypeptide, thereby reducing the potential sideeffects.

1. Production of Nonhuman Antibodies

The present invention features non-human antibodies, for example,antibodies having specificity for the preferred Aβ epitopes of theinvention. Such antibodies can be used in formulating varioustherapeutic compositions of the invention or, preferably, providecomplementarity determining regions for the production of humanized orchimeric antibodies (described in detail below). The production ofnon-human monoclonal antibodies, e.g., murine, guinea pig, primate,rabbit or rat, can be accomplished by, for example, immunizing theanimal with A. A longer polypeptide comprising Aβ or an immunogenicfragment of Aβ or anti-idiotypic antibodies to an antibody to Aβ canalso be used. See Harlow & Lane, supra, incorporated by reference forall purposes). Such an immunogen can be obtained from a natural source,by peptide synthesis or by recombinant expression. Optionally, theimmunogen can be administered fused or otherwise complexed with acarrier protein, as described below. Optionally, the immunogen can beadministered with an adjuvant. The term “adjuvant” refers to a compoundthat when administered in conjunction with an antigen augments theimmune response to the antigen, but when administered alone does notgenerate an immune response to the antigen. Adjuvants can augment animmune response by several mechanisms including lymphocyte recruitment,stimulation of B and/or T cells, and stimulation of macrophages. Severaltypes of adjuvant can be used as described below. Complete Freund'sadjuvant followed by incomplete adjuvant is preferred for immunizationof laboratory animals.

Rabbits or guinea pigs are typically used for making polyclonalantibodies. Exemplary preparation of polyclonal antibodies, e.g., forpassive protection, can be performed as follows. 125 non-transgenic miceare immunized with 100 μg Aβ1-42, plus CFA/IFA adjuvant, and euthanizedat 4-5 months. Blood is collected from immunized mice. IgG is separatedfrom other blood components. Antibody specific for the immunogen may bepartially purified by affinity chromatography. An average of about 0.5-1mg of immunogen-specific antibody is obtained per mouse, giving a totalof 60-120 mg.

Mice are typically used for making monoclonal antibodies. Monoclonalscan be prepared against a fragment by injecting the fragment or longerform of Aβ into a mouse, preparing hybridomas and screening thehybridomas for an antibody that specifically binds to Aβ. Optionally,antibodies are screened for binding to a specific region or desiredfragment of Aβ without binding to other nonoverlapping fragments of Aβ.The latter screening can be accomplished by determining binding of anantibody to a collection of deletion mutants of an Aβ peptide anddetermining which deletion mutants bind to the antibody. Binding can beassessed, for example, by Western blot or ELISA. The smallest fragmentto show specific binding to the antibody defines the epitope of theantibody. Alternatively, epitope specificity can be determined by acompetition assay is which a test and reference antibody compete forbinding to Aβ. If the test and reference antibodies compete, then theybind to the same epitope or epitopes sufficiently proximal such thatbinding of one antibody interferes with binding of the other. Thepreferred isotype for such antibodies is mouse isotype IgG2a orequivalent isotype in other species. Mouse isotype IgG2a is theequivalent of human isotype IgG1 (e.g., human IgG1).

2. Chimeric and Humanized Antibodies

The present invention also features chimeric and/or humanized antibodies(i.e., chimeric and/or humanized immunoglobulins) specific for betaamyloid peptide. Chimeric and/or humanized antibodies have the same orsimilar binding specificity and affinity as a mouse or other nonhumanantibody that provides the starting material for construction of achimeric or humanized antibody.

A. Production of Chimeric Antibodies

The term “chimeric antibody” refers to an antibody whose light and heavychain genes have been constructed, typically by genetic engineering,from immunoglobulin gene segments belonging to different species. Forexample, the variable (V) segments of the genes from a mouse monoclonalantibody may be joined to human constant (C) segments, such as IgG1 andIgG4. Human isotype IgG1 is preferred. A typical chimeric antibody isthus a hybrid protein consisting of the V or antigen-binding domain froma mouse antibody and the C or effector domain from a human antibody.

B. Production of Humanized Antibodies

The term “humanized antibody” refers to an antibody comprising at leastone chain comprising variable region framework residues substantiallyfrom a human antibody chain (referred to as the acceptor immunoglobulinor antibody) and at least one complementarity determining regionsubstantially from a mouse antibody, (referred to as the donorimmunoglobulin or antibody). See, Queen et al., Proc. Natl. Acad. Sci.USA 86:10029-10033 (1989), U.S. Pat. No. 5,530,101, U.S. Pat. No.5,585,089, U.S. Pat. No. 5,693,761, U.S. Pat. No. 5,693,762, Selick etal., WO 90/07861, and Winter, U.S. Pat. No. 5,225,539 (incorporated byreference in their entirety for all purposes). The constant region(s),if present, are also substantially or entirely from a humanimmunoglobulin.

The substitution of mouse CDRs into a human variable domain framework ismost likely to result in retention of their correct spatial orientationif the human variable domain framework adopts the same or similarconformation to the mouse variable framework from which the CDRsoriginated. This is achieved by obtaining the human variable domainsfrom human antibodies whose framework sequences exhibit a high degree ofsequence identity with the murine variable framework domains from whichthe CDRs were derived. The heavy and light chain variable frameworkregions can be derived from the same or different human antibodysequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies or can be consensus sequences ofseveral human antibodies. See Kettleborough et al., Protein Engineering4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993) andCarter et al., WO 92/22653.

Having identified the complementarity determining regions of the murinedonor immunoglobulin and appropriate human acceptor immunoglobulins, thenext step is to determine which, if any, residues from these componentsshould be substituted to optimize the properties of the resultinghumanized antibody. In general, substitution of human amino acidresidues with murine should be minimized, because introduction of murineresidues increases the risk of the antibody eliciting ahuman-anti-mouse-antibody (HAMA) response in humans. Art-recognizedmethods of determining immune response can be performed to monitor aHAMA response in a particular patient or during clinical trials.Patients administered humanized antibodies can be given animmunogenicity assessment at the beginning and throughout theadministration of said therapy. The HAMA response is measured, forexample, by detecting antibodies to the humanized therapeutic reagent,in serum samples from the patient using a method known to one in theart, including surface plasmon resonance technology (BIACORE) and/orsolid-phase ELISA analysis.

Certain amino acids from the human variable region framework residuesare selected for substitution based on their possible influence on CDRconformation and/or binding to antigen. The unnatural juxtaposition ofmurine CDR regions with human variable framework region can result inunnatural conformational restraints, which, unless corrected bysubstitution of certain amino acid residues, lead to loss of bindingaffinity.

The selection of amino acid residues for substitution is determined, inpart, by computer modeling. Computer hardware and software are describedherein for producing three-dimensional images of immunoglobulinmolecules. In general, molecular models are produced starting fromsolved structures for immunoglobulin chains or domains thereof. Thechains to be modeled are compared for amino acid sequence similaritywith chains or domains of solved three-dimensional structures, and thechains or domains showing the greatest sequence similarity is/areselected as starting points for construction of the molecular model.Chains or domains sharing at least 50% sequence identity are selectedfor modeling, and preferably those sharing at least 60%, 70%, 80%, 90%sequence identity or more are selected for modeling. The solved startingstructures are modified to allow for differences between the actualamino acids in the immunoglobulin chains or domains being modeled, andthose in the starting structure. The modified structures are thenassembled into a composite immunoglobulin. Finally, the model is refinedby energy minimization and by verifying that all atoms are withinappropriate distances from one another and that bond lengths and anglesare within chemically acceptable limits.

The selection of amino acid residues for substitution can also bedetermined, in part, by examination of the characteristics of the aminoacids at particular locations, or empirical observation of the effectsof substitution or mutagenesis of particular amino acids. For example,when an amino acid differs between a murine variable region frameworkresidue and a selected human variable region framework residue, thehuman framework amino acid should usually be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

(1) noncovalently binds antigen directly,

(2) is adjacent to a CDR region,

(3) otherwise interacts with a CDR region (e.g., is within about 3-6 Åof a CDR region as determined by computer modeling), or

(4) participates in the VL-VH interface.

Residues which “noncovalently bind antigen directly” include amino acidsin positions in framework regions which are have a good probability ofdirectly interacting with amino acids on the antigen according toestablished chemical forces, for example, by hydrogen bonding, Van derWaals forces, hydrophobic interactions, and the like.

CDR and framework regions are as defined by Kabat et al. or Chothia etal., supra. When framework residues, as defined by Kabat et al., supra,constitute structural loop residues as defined by Chothia et al., supra,the amino acids present in the mouse antibody may be selected forsubstitution into the humanized antibody. Residues which are “adjacentto a CDR region” include amino acid residues in positions immediatelyadjacent to one or more of the CDRs in the primary sequence of thehumanized immunoglobulin chain, for example, in positions immediatelyadjacent to a CDR as defined by Kabat, or a CDR as defined by Chothia(See e.g., Chothia and Lesk J M B 196:901 (1987)). These amino acids areparticularly likely to interact with the amino acids in the CDRs and, ifchosen from the acceptor, to distort the donor CDRs and reduce affinity.Moreover, the adjacent amino acids may interact directly with theantigen (Amit et al., Science, 233:747 (1986), which is incorporatedherein by reference) and selecting these amino acids from the donor maybe desirable to keep all the antigen contacts that provide affinity inthe original antibody.

Residues that “otherwise interact with a CDR region” include those thatare determined by secondary structural analysis to be in a spatialorientation sufficient to affect a CDR region. In one embodiment,residues that “otherwise interact with a CDR region” are identified byanalyzing a three-dimensional model of the donor immunoglobulin (e.g., acomputer-generated model). A three-dimensional model, typically of theoriginal donor antibody, shows that certain amino acids outside of theCDRs are close to the CDRs and have a good probability of interactingwith amino acids in the CDRs by hydrogen bonding, Van der Waals forces,hydrophobic interactions, etc. At those amino acid positions, the donorimmunoglobulin amino acid rather than the acceptor immunoglobulin aminoacid may be selected. Amino acids according to this criterion willgenerally have a side chain atom within about 3 angstrom units (A) ofsome atom in the CDRs and must contain an atom that could interact withthe CDR atoms according to established chemical forces, such as thoselisted above.

In the case of atoms that may form a hydrogen bond, the 3 Å is measuredbetween their nuclei, but for atoms that do not form a bond, the 3 Å ismeasured between their Van der Waals surfaces. Hence, in the lattercase, the nuclei must be within about 6 Å (3 Å plus the sum of the Vander Waals radii) for the atoms to be considered capable of interacting.In many cases the nuclei will be from 4 or 5 to 6 Å apart. Indetermining whether an amino acid can interact with the CDRs, it ispreferred not to consider the last 8 amino acids of heavy chain CDR 2 aspart of the CDRs, because from the viewpoint of structure, these 8 aminoacids behave more as part of the framework.

Amino acids that are capable of interacting with amino acids in theCDRs, may be identified in yet another way. The solvent accessiblesurface area of each framework amino acid is calculated in two ways: (1)in the intact antibody, and (2) in a hypothetical molecule consisting ofthe antibody with its CDRs removed. A significant difference betweenthese numbers of about 10 square angstroms or more shows that access ofthe framework amino acid to solvent is at least partly blocked by theCDRs, and therefore that the amino acid is making contact with the CDRs.Solvent accessible surface area of an amino acid may be calculated basedon a three-dimensional model of an antibody, using algorithms known inthe art (e.g., Connolly, J. Appl. Cryst. 16:548 (1983) and Lee andRichards, J. Mol. Biol. 55:379 (1971), both of which are incorporatedherein by reference). Framework amino acids may also occasionallyinteract with the CDRs indirectly, by affecting the conformation ofanother framework amino acid that in turn contacts the CDRs.

The amino acids at several positions in the framework are known to becapable of interacting with the CDRs in many antibodies (Chothia andLesk, supra, Chothia et al., supra and Tramontano et al., J. Mol. Biol.215:175 (1990), all of which are incorporated herein by reference).Notably, the amino acids at positions 2, 48, 64 and 71 of the lightchain and 26-30, 71 and 94 of the heavy chain (numbering according toKabat) are known to be capable of interacting with the CDRs in manyantibodies. The amino acids at positions 35 in the light chain and 93and 103 in the heavy chain are also likely to interact with the CDRs. Atall these numbered positions, choice of the donor amino acid rather thanthe acceptor amino acid (when they differ) to be in the humanizedimmunoglobulin is preferred. On the other hand, certain residues capableof interacting with the CDR region, such as the first 5 amino acids ofthe light chain, may sometimes be chosen from the acceptorimmunoglobulin without loss of affinity in the humanized immunoglobulin.

Residues which “participate in the VL-VH interface” or “packingresidues” include those residues at the interface between VL and VH asdefined, for example, by Novotny and Haber, Proc. Natl. Acad. Sci. USA,82:4592-66 (1985) or Chothia et al, supra. Generally, unusual packingresidues should be retained in the humanized antibody if they differfrom those in the human frameworks.

In general, one or more of the amino acids fulfilling the above criteriais substituted. In some embodiments, all or most of the amino acidsfulfilling the above criteria are substituted. Occasionally, there issome ambiguity about whether a particular amino acid meets the abovecriteria, and alternative variant immunoglobulins are produced, one ofwhich has that particular substitution, the other of which does not.Alternative variant immunoglobulins so produced can be tested in any ofthe assays described herein for the desired activity, and the preferredimmunoglobulin selected.

Usually the CDR regions in humanized antibodies are substantiallyidentical, and more usually, identical to the corresponding CDR regionsof the donor antibody. Although not usually desirable, it is sometimespossible to make one or more conservative amino acid substitutions ofCDR residues without appreciably affecting the binding affinity of theresulting humanized immunoglobulin. By conservative substitutions isintended combinations such as gly, ala; val, ile, leu; asp, glu; asn,gln; ser, thr; lys, arg; and phe, tyr.

Additional candidates for substitution are acceptor human frameworkamino acids that are unusual or “rare” for a human immunoglobulin atthat position. These amino acids can be substituted with amino acidsfrom the equivalent position of the mouse donor antibody or from theequivalent positions of more typical human immunoglobulins. For example,substitution may be desirable when the amino acid in a human frameworkregion of the acceptor immunoglobulin is rare for that position and thecorresponding amino acid in the donor immunoglobulin is common for thatposition in human immunoglobulin sequences; or when the amino acid inthe acceptor immunoglobulin is rare for that position and thecorresponding amino acid in the donor immunoglobulin is also rare,relative to other human sequences. These criteria help ensure that anatypical amino acid in the human framework does not disrupt the antibodystructure. Moreover, by replacing an unusual human acceptor amino acidwith an amino acid from the donor antibody that happens to be typicalfor human antibodies, the humanized antibody may be made lessimmunogenic.

The term “rare”, as used herein, indicates an amino acid occurring atthat position in less than about 20% but usually less than about 10% ofsequences in a representative sample of sequences, and the term“common”, as used herein, indicates an amino acid occurring in more thanabout 25% but usually more than about 50% of sequences in arepresentative sample. For example, all human light and heavy chainvariable region sequences are respectively grouped into “subgroups” ofsequences that are especially homologous to each other and have the sameamino acids at certain critical positions (Kabat et al., supra). Whendeciding whether an amino acid in a human acceptor sequence is “rare” or“common” among human sequences, it will often be preferable to consideronly those human sequences in the same subgroup as the acceptorsequence.

Additional candidates for substitution are acceptor human frameworkamino acids that would be identified as part of a CDR region under thealternative definition proposed by Chothia et al., supra. Additionalcandidates for substitution are acceptor human framework amino acidsthat would be identified as part of a CDR region under the AbM and/orcontact definitions.

Additional candidates for substitution are acceptor framework residuesthat correspond to a rare or unusual donor framework residue. Rare orunusual donor framework residues are those that are rare or unusual (asdefined herein) for murine antibodies at that position. For murineantibodies, the subgroup can be determined according to Kabat andresidue positions identified which differ from the consensus. Thesedonor specific differences may point to somatic mutations in the murinesequence which enhance activity. Unusual residues that are predicted toaffect binding are retained, whereas residues predicted to beunimportant for binding can be substituted.

Additional candidates for substitution are non-germline residuesoccurring in an acceptor framework region. For example, when an acceptorantibody chain (i.e., a human antibody chain sharing significantsequence identity with the donor antibody chain) is aligned to agermline antibody chain (likewise sharing significant sequence identitywith the donor chain), residues not matching between acceptor chainframework and the germline chain framework can be substituted withcorresponding residues from the germline sequence.

Other than the specific amino acid substitutions discussed above, theframework regions of humanized immunoglobulins are usually substantiallyidentical, and more usually, identical to the framework regions of thehuman antibodies from which they were derived. Of course, many of theamino acids in the framework region make little or no directcontribution to the specificity or affinity of an antibody. Thus, manyindividual conservative substitutions of framework residues can betolerated without appreciable change of the specificity or affinity ofthe resulting humanized immunoglobulin. Thus, in one embodiment thevariable framework region of the humanized immunoglobulin shares atleast 85% sequence identity to a human variable framework regionsequence or consensus of such sequences. In another embodiment, thevariable framework region of the humanized immunoglobulin shares atleast 90%, preferably 95%, more preferably 96%, 97%, 98% or 99% sequenceidentity to a human variable framework region sequence or consensus ofsuch sequences. In general, however, such substitutions are undesirable.

The humanized antibodies preferably exhibit a specific binding affinityfor antigen of at least 10⁷, 10⁸, 10⁹ or 10¹⁰ M⁻¹. Usually the upperlimit of binding affinity of the humanized antibodies for antigen iswithin a factor of three, four or five of that of the donorimmunoglobulin. Often the lower limit of binding affinity is also withina factor of three, four or five of that of donor immunoglobulin.Alternatively, the binding affinity can be compared to that of ahumanized antibody having no substitutions (e.g., an antibody havingdonor CDRs and acceptor FRs, but no FR substitutions). In suchinstances, the binding of the optimized antibody (with substitutions) ispreferably at least two- to three-fold greater, or three- to four-foldgreater, than that of the unsubstituted antibody. For makingcomparisons, activity of the various antibodies can be determined, forexample, by BIACORE (i.e., surface plasmon resonance using unlabelledreagents) or competitive binding assays.

C. Production of Humanized 12B4 Antibodies

A preferred embodiment of the present invention features a humanizedantibody to the N-terminus of Aβ, in particular, for use in thetherapeutic and/or diagnostic methodologies described herein. Aparticularly preferred starting material for production of humanizedantibodies is 12B4. 12B4 is specific for the N-terminus of Aβ and hasbeen shown to mediate phagocytosis (e.g., induce phagocytosis) ofamyloid plaque. The cloning and sequencing of cDNA encoding the 12B4antibody heavy and light chain variable regions is described in ExampleI.

Suitable human acceptor antibody sequences are identified by computercomparisons of the amino acid sequences of the mouse variable regionswith the sequences of known human antibodies. The comparison isperformed separately for heavy and light chains but the principles aresimilar for each. In particular, variable domains from human antibodieswhose framework sequences exhibit a high degree of sequence identitywith the murine VL and VH framework regions were identified by query ofthe Kabat Database using NCBI BLAST (publicly accessible through theNational Institutes of Health NCBI internet server) with the respectivemurine framework sequences. In one embodiment, acceptor sequencessharing greater that 50% sequence identity with murine donor sequencesare selected. Preferably, acceptor antibody sequences sharing 60%, 70%,80%, 90% or more are selected.

A computer comparison of 12B4 revealed that the 12B4 light chain showsthe greatest sequence identity to human light chains of subtype kappaII, and that the 12B4 heavy chain shows greatest sequence identity tohuman heavy chains of subtype II, as defined by Kabat et al., supra.Thus, light and heavy human framework regions are preferably derivedfrom human antibodies of these subtypes, or from consensus sequences ofsuch subtypes. The preferred light chain human variable regions showinggreatest sequence identity to the corresponding region from 12B4 arefrom an antibody having Kabat ID Number 005036. The preferred heavychain human variable regions showing greatest sequence identity to thecorresponding region from 12B4 are from an antibody having Kabat IDNumber 000333, an antibody having Genbank Accession No. AAB35009, and anantibody having Genbank Accession No. AAD53816, with the antibody havingKabat ID Number 000333 being more preferred.

Residues are next selected for substitution, as follows. When an aminoacid differs between a 12B4 variable framework region and an equivalenthuman variable framework region, the human framework amino acid shouldusually be substituted by the equivalent mouse amino acid if it isreasonably expected that the amino acid:

(1) noncovalently binds antigen directly,

(2) is adjacent to a CDR region, is part of a CDR region under thealternative definition proposed by Chothia et al., supra, or otherwiseinteracts with a CDR region (e.g., is within about 3 A of a CDR region),or

(3) participates in the VL-VH interface.

Computer modeling of the 12B4 antibody heavy and light chain variableregions, and humanization of the 12B4 antibody is described in ExampleV. Briefly, a three-dimensional model is generated based on the closestsolved murine antibody structures for the heavy and light chains. Themodel is further refined by a series of energy minimization steps torelieve unfavorable atomic contacts and optimize electrostatic and vander Walls interactions.

Three-dimensional structural information for the antibodies describedherein is publicly available, for example, from the ResearchCollaboratory for Structural Bioinformatics' Protein Data Bank (PDB).The PDB is freely accessible via the World Wide Web internet and isdescribed by Berman et al. (2000) Nucleic Acids Research, 28:235.Computer modeling allows for the identification of CDR-interactingresidues. The computer model of the structure of 12B4 can in turn serveas a starting point for predicting the three-dimensional structure of anantibody containing the 12B4 complementarity determining regionssubstituted in human framework structures. Additional models can beconstructed representing the structure as further amino acidsubstitutions are introduced.

In general, substitution of one, most or all of the amino acidsfulfilling the above criteria is desirable. Accordingly, the humanizedantibodies of the present invention will usually contain a substitutionof a human light chain framework residue with a corresponding 12B4residue in at least 1, 2, 3 or more of the chosen positions. Thehumanized antibodies also usually contain a substitution of a humanheavy chain framework residue with a corresponding 12B4 residue in atleast 1, 2, 3 or more of the chosen positions.

Occasionally, however, there is some ambiguity about whether aparticular amino acid meets the above criteria, and alternative variantimmunoglobulins are produced, one of which has that particularsubstitution, the other of which does not. In instances wheresubstitution with a murine residue would introduce a residue that israre in human immunoglobulins at a particular position, it may bedesirable to test the antibody for activity with or without theparticular substitution. If activity (e.g., binding affinity and/orbinding specificity) is about the same with or without the substitution,the antibody without substitution may be preferred, as it would beexpected to elicit less of a HAMA response, as described herein.

Other candidates for substitution are acceptor human framework aminoacids that are unusual for a human immunoglobulin at that position.These amino acids can be substituted with amino acids from theequivalent position of more typical human immunoglobulins.Alternatively, amino acids from equivalent positions in the mouse 12B4can be introduced into the human framework regions when such amino acidsare typical of human immunoglobulin at the equivalent positions.

Other candidates for substitution are non-germline residues occurring ina framework region. By performing a computer comparison of 12B4 withknown germline sequences, germline sequences with the greatest degree ofsequence identity to the heavy or light chain can be identified.Alignment of the framework region and the germline sequence will revealwhich residues may be selected for substitution with correspondinggermline residues. Residues not matching between a selected light chainacceptor framework and one of these germline sequences could be selectedfor substitution with the corresponding germline residue.

Table 1 summarizes the sequence analysis of the 12B4 VH and VL regions.Additional mouse and human structures that can be used for computermodeling of the 12B4 antibody and additional human antibodies are setforth as well as germline sequences that can be used in selecting aminoacid substitutions. Rare mouse residues are also set forth in Table 1.Rare mouse residues are identified by comparing the donor VL and/or VHsequences with the sequences of other members of the subgroup to whichthe donor VL and/or VH sequences belong (according to Kabat) andidentifying the residue positions which differ from the consensus. Thesedonor specific differences may point to somatic mutations which enhanceactivity. Unusual or rare residues close to the binding site maypossibly contact the antigen, making it desirable to retain the mouseresidue. However, if the unusual mouse residue is not important forbinding, use of the corresponding acceptor residue is preferred as themouse residue may create immunogenic neoepitopes in the humanizedantibody. In the situation where an unusual residue in the donorsequence is actually a common residue in the corresponding acceptorsequence, the preferred residue is clearly the acceptor residue.

TABLE 1 Summary of 12B4 V region sequence Chain VL VH Mouse Subgroup IIIb Human Subgroup II II Rare amino acids (% K107 (0.542%) T3, I11, L12,F24, frequency) S41, N75, D83, A85 Chothia canonical class L1: ~classH1: class 3 [1ggi] 4[1rmf] H2: ~class 1 L2: class 1[1lmk] L3: class1[1tet] Closest mouse MAb solved 2PCP (2.2 Å) 1ETZ (2.6 Å) structureHomology with modeling 94% 80% template Human Framework seq KABID 0050361-KABID 000333 2-AAB35009/1F7 3-AAD53816 Germline ref for Hu FrA3/x12690 & 1: VH4-39/AB019439/ A19/X63397 BAA75036.1 2: VH2-5/AB019440/BAA75057.1

Kabat ID sequences referenced herein are publicly available, forexample, from the Northwestern University Biomedical EngineeringDepartment's Kabat Database of Sequences of Proteins of ImmunologicalInterest. Three-dimensional structural information for antibodiesdescribed herein is publicly available, for example, from the ResearchCollaboratory for Structural Bioinformatics' Protein Data Bank (PDB).The PDB is freely accessible via the World Wide Web internet and isdescribed by Berman et al. (2000) Nucleic Acids Research, p 235-242.Germline gene sequences referenced herein are publicly available, forexample, from the National Center for Biotechnology Information (NCBI)database of sequences in collections of Igh, Ig kappa and Ig lambdagermline V genes (as a division of the National Library of Medicine(NLM) at the National Institutes of Health (NIH)). Homology searching ofthe NCBI “Ig Germline Genes” database is provided by IgG BLAST™.

In a preferred embodiment, a humanized antibody of the present inventioncontains (i) a light chain comprising a variable domain comprisingmurine 12B4 VL CDRs and a human acceptor framework, the framework havingat least one, residue substituted with the corresponding 12B4 residueand (ii) a heavy chain comprising 12B4 VH CDRs and a human acceptorframework, the framework having at least one, preferably two, three,four, five, six, seven, eight, or nine residues substituted with thecorresponding 12B4 residue, and, optionally, at least one, preferablytwo or three residues substituted with a corresponding human germlineresidue.

In another preferred embodiment, a humanized antibody of the presentinvention has structural features, as described herein, and further hasat least one (preferably two, three, four or all) of the followingactivities: (1) binds soluble Aβ; (2) binds aggregated Aβ1-42 (e.g., asdetermined by ELISA); (3) binds Aβ in plaques (e.g., staining of ADand/or PDAPP plaques); (4) binds Aβ with two- to three-fold higherbinding affinity as compared to chimeric 12B4 (e.g., 12B4 having murinevariable region sequences and human constant region sequences); (5)mediates phagocytosis of Aβ (e.g., in an ex vivo phagocytosis assay, asdescribed herein); and (6) crosses the blood-brain barrier (e.g.,demonstrates short-term brain localization, for example, in a PDAPPanimal model, as described herein).

In another preferred embodiment, a humanized antibody of the presentinvention has structural features, as described herein, binds Aβ in amanner or with an affinity sufficient to elicit at least one of thefollowing in vivo effects: (1) reduce Aβ plaque burden; (2) preventplaque formation; (3) reduce levels of soluble Aβ; (4) reduce theneuritic pathology associated with an amyloidogenic disorder; (5) lessenor ameliorate at least one physiological symptom associated with anamyloidogenic disorder; and/or (6) improve cognitive function.

In another preferred embodiment, a humanized antibody of the presentinvention has structural features, as described herein, and specificallybinds to an epitope comprising residues 3-7 of Aβ.

In another preferred embodiment, a humanized antibody of the presentinvention has structural features, as described herein, binds to anN-terminal epitope within Aβ (e.g., binds to an epitope within aminoacids 3-7 of Aβ), and is capable of reducing (1) Aβ peptide levels; (2)Aβ plaque burden; and (3) the neuritic burden or neuritic dystrophyassociated with an amyloidogenic disorder.

The activities described above can be determined utilizing any one of avariety of assays described herein or in the art (e.g., binding assays,phagocytosis assays, etc.). Activities can be assayed either in vivo(e.g., using labeled assay components and/or imaging techniques) or invitro (e.g., using samples or specimens derived from a subject).Activities can be assayed either directly or indirectly. In certainpreferred embodiments, neurological endpoints (e.g., amyloid burden,neuritic burden, etc) are assayed. Such endpoints can be assayed inliving subjects (e.g., in animal models of Alzheimer's disease or inhuman subjects, for example, undergoing immunotherapy) usingnon-invasive detection methodologies. Alternatively, such endpoints canbe assayed in subjects post mortem. Assaying such endpoints in animalmodels and/or in human subjects post mortem is useful in assessing theeffectiveness of various agents (e.g., humanized antibodies) to beutilized in similar immunotherapeutic applications. In other preferredembodiments, behavioral or neurological parameters can be assessed asindicators of the above neuropathological activities or endpoints.

3. Production of Variable Regions

Having conceptually selected the CDR and framework components ofhumanized immunoglobulins, a variety of methods are available forproducing such immunoglobulins. Because of the degeneracy of the code, avariety of nucleic acid sequences will encode each immunoglobulin aminoacid sequence. The desired nucleic acid sequences can be produced by denovo solid-phase DNA synthesis or by PCR mutagenesis of an earlierprepared variant of the desired polynucleotide. Oligonucleotide-mediatedmutagenesis is a preferred method for preparing substitution, deletionand insertion variants of target polypeptide DNA. See Adelman et al.,DNA 2:183 (1983). Briefly, the target polypeptide DNA is altered byhybridizing an oligonucleotide encoding the desired mutation to asingle-stranded DNA template. After hybridization, a DNA polymerase isused to synthesize an entire second complementary strand of the templatethat incorporates the oligonucleotide primer, and encodes the selectedalteration in the target polypeptide DNA.

4. Selection of Constant Regions

The variable segments of antibodies produced as described supra (e.g.,the heavy and light chain variable regions of chimeric or humanizedantibodies) are typically linked to at least a portion of animmunoglobulin constant region (Fc region), typically that of a humanimmunoglobulin. Human constant region DNA sequences can be isolated inaccordance with well known procedures from a variety of human cells, butpreferably immortalized B cells (see Kabat et al., supra, and Liu etal., WO87/02671) (each of which is incorporated by reference in itsentirety for all purposes). Ordinarily, the antibody will contain bothlight chain and heavy chain constant regions. The heavy chain constantregion usually includes CH1, hinge, CH2, CH3, and CH4 regions. Theantibodies described herein include antibodies having all types ofconstant regions, including IgM, IgG, IgD, IgA and IgE, and any isotype,including IgG1, IgG2, IgG3 and IgG4. When it is desired that theantibody (e.g., humanized antibody) exhibit cytotoxic activity, theconstant domain is usually a complement fixing constant domain and theclass is typically IgG1. Human isotype IgG1 is preferred. Light chainconstant regions can be lambda or kappa. The humanized antibody maycomprise sequences from more than one class or isotype. Antibodies canbe expressed as tetramers containing two light and two heavy chains, asseparate heavy chains, light chains, as Fab, Fab′ F(ab′)2, and Fv, or assingle chain antibodies in which heavy and light chain variable domainsare linked through a spacer.

5. Expression of Recombinant Antibodies

Chimeric and humanized antibodies are typically produced by recombinantexpression. Nucleic acids encoding light and heavy chain variableregions, optionally linked to constant regions, are inserted intoexpression vectors. The light and heavy chains can be cloned in the sameor different expression vectors. The DNA segments encodingimmunoglobulin chains are operably linked to control sequences in theexpression vector(s) that ensure the expression of immunoglobulinpolypeptides. Expression control sequences include, but are not limitedto, promoters (e.g., naturally-associated or heterologous promoters),signal sequences, enhancer elements, and transcription terminationsequences. Preferably, the expression control sequences are eukaryoticpromoter systems in vectors capable of transforming or transfectingeukaryotic host cells. Once the vector has been incorporated into theappropriate host, the host is maintained under conditions suitable forhigh level expression of the nucleotide sequences, and the collectionand purification of the crossreacting antibodies.

These expression vectors are typically replicable in the host organismseither as episomes or as an integral part of the host chromosomal DNA.Commonly, expression vectors contain selection markers (e.g.,ampicillin-resistance, hygromycin-resistance, tetracycline resistance,kanamycin resistance or neomycin resistance) to permit detection ofthose cells transformed with the desired DNA sequences (see, e.g.,Itakura et al., U.S. Pat. No. 4,704,362).

E. coli is one prokaryotic host particularly useful for cloning thepolynucleotides (e.g., DNA sequences) of the present invention. Othermicrobial hosts suitable for use include bacilli, such as Bacillussubtilis, and other enterobacteriaceae, such as Salmonella, Serratia,and various Pseudomonas species. In these prokaryotic hosts, one canalso make expression vectors, which will typically contain expressioncontrol sequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation. Other microbes, such as yeast, are alsouseful for expression.

Saccharomyces is a preferred yeast host, with suitable vectors havingexpression control sequences (e.g., promoters), an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

In addition to microorganisms, mammalian tissue cell culture may also beused to express and produce the polypeptides of the present invention(e.g., polynucleotides encoding immunoglobulins or fragments thereof).See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987).Eukaryotic cells are actually preferred, because a number of suitablehost cell lines capable of secreting heterologous proteins (e.g., intactimmunoglobulins) have been developed in the art, and include CHO celllines, various Cos cell lines, HeLa cells, preferably, myeloma celllines, or transformed B-cells or hybridomas. Preferably, the cells arenonhuman. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, and anenhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, SV40, adenovirus, bovine papilloma virus,cytomegalovirus and the like. See Co et al., J. Immunol. 148:1149(1992).

Alternatively, antibody-coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,Deboer et al., U.S. Pat. No. 5,741,957, Rosen, U.S. Pat. No. 5,304,489,and Meade et al., U.S. Pat. No. 5,849,992). Suitable transgenes includecoding sequences for light and/or heavy chains in operable linkage witha promoter and enhancer from a mammary gland specific gene, such ascasein or beta lactoglobulin.

The vectors containing the polynucleotide sequences of interest (e.g.,the heavy and light chain encoding sequences and expression controlsequences) can be transferred into the host cell by well-known methods,which vary depending on the type of cellular host. For example, calciumchloride transfection is commonly utilized for prokaryotic cells,whereas calcium phosphate treatment, electroporation, lipofection,biolistics or viral-based transfection may be used for other cellularhosts. (See generally Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Press, 2nd ed., 1989) (incorporated byreference in its entirety for all purposes). Other methods used totransform mammalian cells include the use of polybrene, protoplastfusion, liposomes, electroporation, and microinjection (see generally,Sambrook et al., supra). For production of transgenic animals,transgenes can be microinjected into fertilized oocytes, or can beincorporated into the genome of embryonic stem cells, and the nuclei ofsuch cells transferred into enucleated oocytes.

When heavy and light chains are cloned on separate expression vectors,the vectors are co-transfected to obtain expression and assembly ofintact immunoglobulins. Once expressed, the whole antibodies, theirdimers, individual light and heavy chains, or other immunoglobulin formsof the present invention can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns, column chromatography, HPLC purification, gelelectrophoresis and the like (see generally Scopes, Protein Purification(Springer-Verlag, N.Y., (1982)). Substantially pure immunoglobulins ofat least about 90 to 95% homogeneity are preferred, and 98 to 99% ormore homogeneity most preferred, for pharmaceutical uses.

6. Antibody Fragments

Also contemplated within the scope of the instant invention are antibodyfragments. In one embodiment, fragments of non-human, and/or chimericantibodies are provided. In another embodiment, fragments of humanizedantibodies are provided. Typically, these fragments exhibit specificbinding to antigen with an affinity of at least 10⁷, and more typically10⁸ or 10⁹M⁻¹. Humanized antibody fragments include separate heavychains, light chains, Fab, Fab′, F(ab′)2, Fabc, and Fv. Fragments areproduced by recombinant DNA techniques, or by enzymatic or chemicalseparation of intact immunoglobulins.

7. Testing Antibodies for Therapeutic Efficacy in Animal Models

Groups of 7-9 month old PDAPP mice each are injected with 0.5 mg in PBSof polyclonal anti-Aβ or specific anti-Aβ monoclonal antibodies. Allantibody preparations are purified to have low endotoxin levels.Monoclonals can be prepared against a fragment by injecting the fragmentor longer form of Aβ into a mouse, preparing hybridomas and screeningthe hybridomas for an antibody that specifically binds to a desiredfragment of Aβ without binding to other nonoverlapping fragments of Aβ.

Mice are injected intraperitoneally as needed over a 4 month period tomaintain a circulating antibody concentration measured by ELISA titer ofgreater than 1/1000 defined by ELISA to Aβ42 or other immunogen. Titersare monitored and mice are euthanized at the end of 6 months ofinjections. Histochemistry, Aβ levels and toxicology are performed postmortem. Ten mice are used per group.

8. Screening Antibodies for Clearing Activity

The invention also provides methods of screening an antibody foractivity in clearing an amyloid deposit or any other antigen, orassociated biological entity, for which clearing activity is desired. Toscreen for activity against an amyloid deposit, a tissue sample from abrain of a patient with Alzheimer's disease or an animal model havingcharacteristic Alzheimer's pathology is contacted with phagocytic cellsbearing an Fc receptor, such as microglial cells, and the antibody undertest in a medium in vitro. The phagocytic cells can be a primary cultureor a cell line, and can be of murine (e.g., BV-2 or C8-B4 cells) orhuman origin (e.g., THP-1 cells). In some methods, the components arecombined on a microscope slide to facilitate microscopic monitoring. Insome methods, multiple reactions are performed in parallel in the wellsof a microtiter dish. In such a format, a separate miniature microscopeslide can be mounted in the separate wells, or a nonmicroscopicdetection format, such as ELISA detection of Aβ can be used. Preferably,a series of measurements is made of the amount of amyloid deposit in thein vitro reaction mixture, starting from a baseline value before thereaction has proceeded, and one or more test values during the reaction.The antigen can be detected by staining, for example, with afluorescently labeled antibody to Aβ or other component of amyloidplaques. The antibody used for staining may or may not be the same asthe antibody being tested for clearing activity. A reduction relative tobaseline during the reaction of the amyloid deposits indicates that theantibody under test has clearing activity. Such antibodies are likely tobe useful in preventing or treating Alzheimer's and other amyloidogenicdiseases. Particularly useful antibodies for preventing or treatingAlzheimer's and other amyloidogenic diseases include those capable ofclearing both compact and diffuse amyloid plaques, for example, the 12B4antibody of the instant invention, or chimeric or humanized versionsthereof.

Analogous methods can be used to screen antibodies for activity inclearing other types of biological entities. The assay can be used todetect clearing activity against virtually any kind of biologicalentity. Typically, the biological entity has some role in human oranimal disease. The biological entity can be provided as a tissue sampleor in isolated form. If provided as a tissue sample, the tissue sampleis preferably unfixed to allow ready access to components of the tissuesample and to avoid perturbing the conformation of the componentsincidental to fixing. Examples of tissue samples that can be tested inthis assay include cancerous tissue, precancerous tissue, tissuecontaining benign growths such as warts or moles, tissue infected withpathogenic microorganisms, tissue infiltrated with inflammatory cells,tissue bearing pathological matrices between cells (e.g., fibrinouspericarditis), tissue bearing aberrant antigens, and scar tissue.Examples of isolated biological entities that can be used include Aβ,viral antigens or viruses, proteoglycans, antigens of other pathogenicmicroorganisms, tumor antigens, and adhesion molecules. Such antigenscan be obtained from natural sources, recombinant expression or chemicalsynthesis, among other means. The tissue sample or isolated biologicalentity is contacted with phagocytic cells bearing Fc receptors, such asmonocytes or microglial cells, and an antibody to be tested in a medium.The antibody can be directed to the biological entity under test or toan antigen associated with the entity. In the latter situation, theobject is to test whether the biological entity is phagocytosed with theantigen. Usually, although not necessarily, the antibody and biologicalentity (sometimes with an associated antigen), are contacted with eachother before adding the phagocytic cells. The concentration of thebiological entity and/or the associated antigen remaining in the medium,if present, is then monitored. A reduction in the amount orconcentration of antigen or the associated biological entity in themedium indicates the antibody has a clearing response against theantigen and/or associated biological entity in conjunction with thephagocytic cells (see, e.g., Example IV).

9. Chimeric/Humanized Antibodies Having Altered Effector Function

For the above-described antibodies of the invention comprising aconstant region (Fc region), it may also be desirable to alter theeffector function of the molecule. Generally, the effector function ofan antibody resides in the constant or Fc region of the molecule whichcan mediate binding to various effector molecules, e.g., complementproteins or Fc receptors. The binding of complement to the Fc region isimportant, for example, in the opsonization and lysis of cell pathogensand the activation of inflammatory responses. The binding of antibody toFc receptors, for example, on the surface of effector cells can triggera number of important and diverse biological responses including, forexample, engulfment and destruction of antibody-coated pathogens orparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (i.e., antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer of antibodies, and control of immunoglobulin production.

Accordingly, depending on a particular therapeutic or diagnosticapplication, the above-mentioned immune functions, or only selectedimmune functions, may be desirable. By altering the Fc region of theantibody, various aspects of the effector function of the molecule,including enhancing or suppressing various reactions of the immunesystem, with beneficial effects in diagnosis and therapy, are achieved.

The antibodies of the invention can be produced which react only withcertain types of Fc receptors, for example, the antibodies of theinvention can be modified to bind to only certain Fc receptors, or ifdesired, lack Fc receptor binding entirely, by deletion or alteration ofthe Fc receptor binding site located in the Fc region of the antibody.Other desirable alterations of the Fc region of an antibody of theinvention are cataloged below. Typically the Kabat numbering system isused to indicate which amino acid residue(s) of the Fc region (e.g., ofan IgG antibody) are altered (e.g., by amino acid substitution) in orderto achieve a desired change in effector function. The numbering systemis also employed to compare antibodies across species such that adesired effector function observed in, for example, a mouse antibody,can then be systematically engineered into a human, humanized, orchimeric antibody of the invention.

For example, it has been observed that antibodies (e.g., IgG antibodies)can be grouped into those found to exhibit tight, intermediate, or weakbinding to an Fc receptor (e.g., an Fc receptor on human monocytes(FcγRI)). By comparison of the amino-acid sequences in these differentaffinity groups, a monocyte-binding site in the hinge-link region(Leu234-Ser239) has been identified. Moreover, the human FcγRI receptorbinds human IgG1 and mouse IgG2a as a monomer, but the binding of mouseIgG2b is 100-fold weaker. A comparison of the sequence of these proteinsin the hinge-link region shows that the sequence 234 to 238, i.e.,Leu-Leu-Gly-Gly-Pro in the strong binders becomes Leu-Glu-Gly-Gly-Pro inmouse gamma 2b, i.e., weak binders. Accordingly, a corresponding changein a human antibody hinge sequence can be made if reduced FcγI receptorbinding is desired. It is understood that other alterations can be madeto achieve the same or similar results. For example, the affinity ofFcγRI binding can be altered by replacing the specified residue with aresidue having an inappropriate functional group on its sidechain, or byintroducing a charged functional group (e.g., Glu or Asp) or for examplean aromatic non-polar residue (e.g., Phe, Tyr, or Trp).

These changes can be equally applied to the murine, human, and ratsystems given the sequence homology between the differentimmunoglobulins. It has been shown that for human IgG3, which binds tothe human FcγRI receptor, changing Leu 235 to Glu destroys theinteraction of the mutant for the receptor. The binding site for thisreceptor can thus be switched on or switched off by making theappropriate mutation.

Mutations on adjacent or close sites in the hinge link region (e.g.,replacing residues 234, 236 or 237 by Ala) indicate that alterations inresidues 234, 235, 236, and 237 at least affect affinity for the FcγRIreceptor. Accordingly, the antibodies of the invention can also have analtered Fc region with altered binding affinity for FcγRI as comparedwith the unmodified antibody. Such an antibody conveniently has amodification at amino acid residue 234, 235, 236, or 237.

Affinity for other Fc receptors can be altered by a similar approach,for controlling the immune response in different ways.

As a further example, the lytic properties of IgG antibodies followingbinding of the Cl component of complement can be altered.

The first component of the complement system, Cl, comprises threeproteins known as Clq, Clr and Cls which bind tightly together. It hasbeen shown that Clq is responsible for binding of the three proteincomplex to an antibody.

Accordingly, the Clq binding activity of an antibody can be altered byproviding an antibody with an altered CH 2 domain in which at least oneof the amino acid residues 318, 320, and 322 of the heavy chain has beenchanged to a residue having a different side chain. The numbering of theresidues in the heavy chain is that of the EU index (see Kabat et al.,supra). Other suitable alterations for altering, e.g., reducing orabolishing specific Clq-binding to an antibody include changing any oneof residues 318 (Glu), 320 (Lys) and 322 (Lys), to Ala.

Moreover, by making mutations at these residues, it has been shown thatClq binding is retained as long as residue 318 has a hydrogen-bondingside chain and residues 320 and 322 both have a positively charged sidechain.

Clq binding activity can be abolished by replacing any one of the threespecified residues with a residue having an inappropriate functionalityon its side chain. It is not necessary to replace the ionic residuesonly with Ala to abolish Clq binding. It is also possible to use otheralkyl-substituted non-ionic residues, such as Gly, Ile, Leu, or Val, orsuch aromatic non-polar residues as Phe, Tyr, Trp and Pro in place ofany one of the three residues in order to abolish Clq binding. Inaddition, it is also be possible to use such polar non-ionic residues asSer, Thr, Cys, and Met in place of residues 320 and 322, but not 318, inorder to abolish Clq binding activity.

It is also noted that the side chains on ionic or non-ionic polarresidues will be able to form hydrogen bonds in a similar manner to thebonds formed by the Glu residue. Therefore, replacement of the 318 (Glu)residue by a polar residue may modify but not abolish Clq bindingactivity.

It is also known that replacing residue 297 (Asn) with Ala results inremoval of lytic activity while only slightly reducing (about three foldweaker) affinity for Clq. This alteration destroys the glycosylationsite and the presence of carbohydrate that is required for complementactivation. Any other substitution at this site will also destroy theglycosylation site.

The invention also provides an antibody having an altered effectorfunction wherein the antibody has a modified hinge region. The modifiedhinge region may comprise a complete hinge region derived from anantibody of different antibody class or subclass from that of the CH1domain. For example, the constant domain (CH1) of a class IgG antibodycan be attached to a hinge region of a class IgG4 antibody.Alternatively, the new hinge region may comprise part of a natural hingeor a repeating unit in which each unit in the repeat is derived from anatural hinge region. In one example, the natural hinge region isaltered by converting one or more cysteine residues into a neutralresidue, such as alanine, or by converting suitably placed residues intocysteine residues. Such alterations are carried out using art recognizedprotein chemistry and, preferably, genetic engineering techniques, asdescribed herein.

In one embodiment of the invention, the number of cysteine residues inthe hinge region of the antibody is reduced, for example, to onecysteine residue. This modification has the advantage of facilitatingthe assembly of the antibody, for example, bispecific antibody moleculesand antibody molecules wherein the Fc portion has been replaced by aneffector or reporter molecule, since it is only necessary to form asingle disulfide bond. This modification also provides a specific targetfor attaching the hinge region either to another hinge region or to aneffector or reporter molecule, either directly or indirectly, forexample, by chemical means.

Conversely, the number of cysteine residues in the hinge region of theantibody is increased, for example, at least one more than the number ofnormally occurring cysteine residues. Increasing the number of cysteineresidues can be used to stabilize the interactions between adjacenthinges. Another advantage of this modification is that it facilitatesthe use of cysteine thiol groups for attaching effector or reportermolecules to the altered antibody, for example, a radiolabel.

Accordingly, the invention provides for an exchange of hinge regionsbetween antibody classes, in particular, IgG classes, and/or an increaseor decrease in the number of cysteine residues in the hinge region inorder to achieve an altered effector function (see for example U.S. Pat.No. 5,677,425 which is expressly incorporated herein). A determinationof altered antibody effector function is made using the assays describedherein or other art recognized techniques.

Importantly, the resultant antibody can be subjected to one or moreassays to evaluate any change in biological activity compared to thestarting antibody. For example, the ability of the antibody with analtered Fc region to bind complement or Fc receptors can be assessedusing the assays disclosed herein as well as any art recognized assay.

Production of the antibodies of the invention is carried out by anysuitable technique including techniques described herein as well astechniques known to those skilled in the art. For example an appropriateprotein sequence, e.g. forming part of or all of a relevant constantdomain, e.g., Fc region, i.e., CH2, and/or CH3 domain(s), of anantibody, and include appropriately altered residue(s) can besynthesized and then chemically joined into the appropriate place in anantibody molecule.

Preferably, genetic engineering techniques are used for producing analtered antibody. Preferred techniques include, for example, preparingsuitable primers for use in polymerase chain reaction (PCR) such that aDNA sequence which encodes at least part of an IgG heavy chain, e.g., anFc or constant region (e.g., CH2, and/or CH3) is altered, at one or moreresidues. The segment can then be operably linked to the remainingportion of the antibody, e.g., the variable region of the antibody andrequired regulatory elements for expression in a cell.

The present invention also includes vectors used to transform the cellline, vectors used in producing the transforming vectors, cell linestransformed with the transforming vectors, cell lines transformed withpreparative vectors, and methods for their production.

Preferably, the cell line which is transformed to produce the antibodywith an altered Fc region (i.e., of altered effector function) is animmortalized mammalian cell line (e.g., CHO cell).

Although the cell line used to produce the antibody with an altered Fcregion is preferably a mammalian cell line, any other suitable cellline, such as a bacterial cell line or a yeast cell line, mayalternatively be used.

B. Nucleic Acid Encoding Immunologic and Therapeutic Agents

Immune responses against amyloid deposits can also be induced byadministration of nucleic acids encoding antibodies and their componentchains used for passive immunization. Such nucleic acids can be DNA orRNA. A nucleic acid segment encoding an immunogen is typically linked toregulatory elements, such as a promoter and enhancer, that allowexpression of the DNA segment in the intended target cells of a patient.For expression in blood cells, as is desirable for induction of animmune response, promoter and enhancer elements from light or heavychain immunoglobulin genes or the CMV major intermediate early promoterand enhancer are suitable to direct expression. The linked regulatoryelements and coding sequences are often cloned into a vector. Foradministration of double-chain antibodies, the two chains can be clonedin the same or separate vectors.

A number of viral vector systems are available including retroviralsystems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop.3:102-109 (1993)); adenoviral vectors (see, e.g., Bett et al., J. Virol.67:5911 (1993)); adeno-associated virus vectors (see, e.g., Zhou et al.,J. Exp. Med. 179:1867 (1994)), viral vectors from the pox familyincluding vaccinia virus and the avian pox viruses, viral vectors fromthe alpha virus genus such as those derived from Sindbis and SemlikiForest Viruses (see, e.g., Dubensky et al., J. Virol. 70:508 (1996)),Venezuelan equine encephalitis virus (see Johnston et al., U.S. Pat. No.5,643,576) and rhabdoviruses, such as vesicular stomatitis virus (seeRose, U.S. Pat. No. 6,168,943) and papillomaviruses (Ohe et al., HumanGene Therapy 6:325 (1995); Woo et al., WO 94/12629 and Xiao & Brandsma,Nucleic Acids. Res. 24, 2630-2622 (1996)).

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by Eppstein et al., U.S. Pat. No. 5,208,036, Feigner et al.,U.S. Pat. No. 5,264,618, Rose, U.S. Pat. No. 5,279,833, and Epand etal., U.S. Pat. No. 5,283,185. Vectors and DNA encoding an immunogen canalso be adsorbed to or associated with particulate carriers, examples ofwhich include polymethyl methacrylate polymers and polylactides andpoly(lactide-co-glycolides), see, e.g., McGee et al., J. Micro Encap.(1996).

Gene therapy vectors or naked polypeptides (e.g., DNA) can be deliveredin vivo by administration to an individual patient, typically bysystemic administration (e.g., intravenous, intraperitoneal, nasal,gastric, intradermal, intramuscular, subdermal, or intracranialinfusion) or topical application (see e.g., Anderson et al., U.S. Pat.No. 5,399,346). The term “naked polynucleotide” refers to apolynucleotide not complexed with colloidal materials. Nakedpolynucleotides are sometimes cloned in a plasmid vector. Such vectorscan further include facilitating agents such as bupivacine (Weiner etal., U.S. Pat. No. 5,593,972). DNA can also be administered using a genegun. See Xiao & Brandsma, supra. The DNA encoding an immunogen isprecipitated onto the surface of microscopic metal beads. Themicroprojectiles are accelerated with a shock wave or expanding heliumgas, and penetrate tissues to a depth of several cell layers. Forexample, The Accel™ Gene Delivery Device manufactured by Agricetus, Inc.Middleton Wis. is suitable. Alternatively, naked DNA can pass throughskin into the blood stream simply by spotting the DNA onto skin withchemical or mechanical irritation (see Howell et al., WO 95/05853).

In a further variation, vectors encoding immunogens can be delivered tocells ex vivo, such as cells explanted from an individual patient (e.g.,lymphocytes, bone marrow aspirates, tissue biopsy) or universal donorhematopoietic stem cells, followed by reimplantation of the cells into apatient, usually after selection for cells which have incorporated thevector.

II. Prophylactic and Therapeutic Methods

The present invention is directed inter alis to treatment of Alzheimer'sand other amyloidogenic diseases by administration of therapeuticimmunological reagents (e.g., humanized immunoglobulins) to specificepitopes within Aβ to a patient under conditions that generate abeneficial therapeutic response in a patient (e.g., induction ofphagocytosis of Aβ, reduction of plaque burden, inhibition of plaqueformation, reduction of neuritic dystrophy, improving cognitivefunction, and/or reversing, treating or preventing cognitive decline) inthe patient, for example, for the prevention or treatment of anamyloidogenic disease. The invention is also directed to use of thedisclosed immunological reagents (e.g., humanized immunoglobulins) inthe manufacture of a medicament for the treatment or prevention of anamyloidogenic disease.

In one aspect, the invention provides methods of preventing or treatinga disease associated with amyloid deposits of Aβ in the brain of apatient. Such diseases include Alzheimer's disease, Down's syndrome andcognitive impairment. The latter can occur with or without othercharacteristics of an amyloidogenic disease. Some methods of theinvention entail administering an effective dosage of an antibody thatspecifically binds to a component of an amyloid deposit to the patient.Such methods are particularly useful for preventing or treatingAlzheimer's disease in human patients. Exemplary methods entailadministering an effective dosage of an antibody that binds to Aβ.Preferred methods entail administering an effective dosage of anantibody that specifically binds to an epitope within residues 1-10 ofAβ, for example, antibodies that specifically bind to an epitope withinresidues 1-3 of Aβ, antibodies that specifically bind to an epitopewithin residues 1-4 of Aβ, antibodies that specifically bind to anepitope within residues 1-5 of Aβ, antibodies that specifically bind toan epitope within residues 1-6 of Aβ, antibodies that specifically bindto an epitope within residues 1-7 of Aβ, or antibodies that specificallybind to an epitope within residues 3-7 of Aβ. In yet another aspect, theinvention features administering antibodies that bind to an epitopecomprising a free N-terminal residue of Aβ. In yet another aspect, theinvention features administering antibodies that bind to an epitopewithin residues of 1-10 of Aβ wherein residue 1 and/or residue 7 of Aβis aspartic acid. In yet another aspect, the invention featuresadministering antibodies that specifically bind to Aβ peptide withoutbinding to full-length amyloid precursor protein (APP). In yet anotheraspect, the isotype of the antibody is human IgG1.

In yet another aspect, the invention features administering antibodiesthat bind to an amyloid deposit in the patient and induce a clearingresponse against the amyloid deposit. For example, such a clearingresponse can be effected by Fc receptor mediated phagocytosis.

Therapeutic agents of the invention are typically substantially purefrom undesired contaminant. This means that an agent is typically atleast about 50% w/w (weight/weight) purity, as well as beingsubstantially free from interfering proteins and contaminants. Sometimesthe agents are at least about 80% w/w and, more preferably at least 90or about 95% w/w purity. However, using conventional proteinpurification techniques, homogeneous peptides of at least 99% w/w can beobtained.

The methods can be used on both asymptomatic patients and thosecurrently showing symptoms of disease. The antibodies used in suchmethods can be human, humanized, chimeric or nonhuman antibodies, orfragments thereof (e.g., antigen binding fragments) and can bemonoclonal or polyclonal, as described herein. In yet another aspect,the invention features administering antibodies prepared from a humanimmunized with Aβ peptide, which human can be the patient to be treatedwith antibody.

In another aspect, the invention features administering an antibody witha pharmaceutical carrier as a pharmaceutical composition. Alternatively,the antibody can be administered to a patient by administering apolynucleotide encoding at least one antibody chain. The polynucleotideis expressed to produce the antibody chain in the patient. Optionally,the polynucleotide encodes heavy and light chains of the antibody. Thepolynucleotide is expressed to produce the heavy and light chains in thepatient. In exemplary embodiments, the patient is monitored for level ofadministered antibody in the blood of the patient.

The invention thus fulfills a longstanding need for therapeutic regimesfor preventing or ameliorating the neuropathology and, in some patients,the cognitive impairment associated with Alzheimer's disease.

A. Patients Amenable to Treatment

Patients amenable to treatment include individuals at risk of diseasebut not showing symptoms, as well as patients presently showingsymptoms. In the case of Alzheimer's disease, virtually anyone is atrisk of suffering from Alzheimer's disease if he or she lives longenough. Therefore, the present methods can be administeredprophylactically to the general population without the need for anyassessment of the risk of the subject patient. The present methods areespecially useful for individuals who have a known genetic risk ofAlzheimer's disease. Such individuals include those having relatives whohave experienced this disease, and those whose risk is determined byanalysis of genetic or biochemical markers. Genetic markers of risktoward Alzheimer's disease include mutations in the APP gene,particularly mutations at position 717 and positions 670 and 671referred to as the Hardy and Swedish mutations respectively (see Hardy,supra). Other markers of risk are mutations in the presenilin genes, PS1and PS2, and ApoE4, family history of AD, hypercholesterolemia oratherosclerosis. Individuals presently suffering from Alzheimer'sdisease can be recognized from characteristic dementia, as well as thepresence of risk factors described above. In addition, a number ofdiagnostic tests are available for identifying individuals who have AD.These include measurement of CSF tau and Aβ42 levels. Elevated tau anddecreased Aβ42 levels signify the presence of AD. Individuals sufferingfrom Alzheimer's disease can also be diagnosed by ADRDA criteria asdiscussed in the Examples section.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70. Treatment typically entails multipledosages over a period of time. Treatment can be monitored by assayingantibody levels over time. If the response falls, a booster dosage isindicated. In the case of potential Down's syndrome patients, treatmentcan begin antenatally by administering therapeutic agent to the motheror shortly after birth.

B. Treatment Regimes and Dosages

In prophylactic applications, pharmaceutical compositions or medicamentsare administered to a patient susceptible to, or otherwise at risk of,Alzheimer's disease in an amount sufficient to eliminate or reduce therisk, lessen the severity, or delay the outset of the disease, includingbiochemical, histologic and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. In therapeutic applications, compositions ormedicaments are administered to a patient suspected of, or alreadysuffering from such a disease in an amount sufficient to cure, or atleast partially arrest, the symptoms of the disease (biochemical,histologic and/or behavioral), including its complications andintermediate pathological phenotypes in development of the disease.

In some methods, administration of agent reduces or eliminatesmyocognitive impairment in patients that have not yet developedcharacteristic Alzheimer's pathology. An amount adequate to accomplishtherapeutic or prophylactic treatment is defined as a therapeutically-or prophylactically-effective dose. In both prophylactic and therapeuticregimes, agents are usually administered in several dosages until asufficient immune response has been achieved. The term “immune response”or “immunological response” includes the development of a humoral(antibody mediated) and/or a cellular (mediated by antigen-specific Tcells or their secretion products) response directed against an antigenin a recipient subject. Such a response can be an active response, i.e.,induced by administration of immunogen, or a passive response, i.e.,induced by administration of immunoglobulin or antibody or primedT-cells. Typically, the immune response is monitored and repeateddosages are given if the immune response starts to wane.

Effective doses of the compositions of the present invention, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnon-human mammals including transgenic mammals can also be treated.Treatment dosages need to be titrated to optimize safety and efficacy.

For passive immunization with an antibody, the dosage ranges from about0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg (e.g., 0.02 mg/kg,0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.), of the hostbody weight. For example dosages can be 1 mg/kg body weight or 10 mg/kgbody weight or within the range of 1-10 mg/kg, preferably at least 1mg/kg. Doses intermediate in the above ranges are also intended to bewithin the scope of the invention. Subjects can be administered suchdoses daily, on alternative days, weekly or according to any otherschedule determined by empirical analysis. An exemplary treatmententails administration in multiple dosages over a prolonged period, forexample, of at least six months. Additional exemplary treatment regimesentail administration once per every two weeks or once a month or onceevery 3 to 6 months. Exemplary dosage schedules include 1-10 mg/kg or 15mg/kg on consecutive days, 30 mg/kg on alternate days or 60 mg/kgweekly. In some methods, two or more monoclonal antibodies withdifferent binding specificities are administered simultaneously, inwhich case the dosage of each antibody administered falls within theranges indicated.

Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. Intervals canalso be irregular as indicated by measuring blood levels of antibody toAβ in the patient. In some methods, dosage is adjusted to achieve aplasma antibody concentration of 1-1000 μg/ml and in some methods 25-300μg/ml. Alternatively, antibody can be administered as a sustainedrelease formulation, in which case less frequent administration isrequired. Dosage and frequency vary depending on the half-life of theantibody in the patient. In general, humanized antibodies show thelongest half-life, followed by chimeric antibodies and nonhumanantibodies.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, compositions containing the present antibodies or acocktail thereof are administered to a patient not already in thedisease state to enhance the patient's resistance. Such an amount isdefined to be a “prophylactic effective dose.” In this use, the preciseamounts again depend upon the patient's state of health and generalimmunity, but generally range from 0.1 to 25 mg per dose, especially 0.5to 2.5 mg per dose. A relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives.

In therapeutic applications, a relatively high dosage (e.g., from about1 to 200 mg of antibody per dose, with dosages of from 5 to 25 mg beingmore commonly used) at relatively short intervals is sometimes requireduntil progression of the disease is reduced or terminated, andpreferably until the patient shows partial or complete amelioration ofsymptoms of disease. Thereafter, the patent can be administered aprophylactic regime.

Doses for nucleic acids encoding antibodies range from about 10 ng to 1g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Dosesfor infectious viral vectors vary from 10-100, or more, virions perdose.

Therapeutic agents can be administered by parenteral, topical,intravenous, oral, subcutaneous, intraarterial, intracranial,intraperitoneal, intranasal or intramuscular means for prophylacticand/or therapeutic treatment. The most typical route of administrationof an immunogenic agent is subcutaneous although other routes can beequally effective. The next most common route is intramuscularinjection. This type of injection is most typically performed in the armor leg muscles. In some methods, agents are injected directly into aparticular tissue where deposits have accumulated, for exampleintracranial injection. Intramuscular injection or intravenous infusionare preferred for administration of antibody. In some methods,particular therapeutic antibodies are injected directly into thecranium. In some methods, antibodies are administered as a sustainedrelease composition or device, such as a Medipad™ device.

Agents of the invention can optionally be administered in combinationwith other agents that are at least partly effective in treatment ofamyloidogenic disease. In the case of Alzheimer's and Down's syndrome,in which amyloid deposits occur in the brain, agents of the inventioncan also be administered in conjunction with other agents that increasepassage of the agents of the invention across the blood-brain barrier.Agents of the invention can also be administered in combination withother agents that enhance access of the therapeutic agent to a targetcell or tissue, for example, liposomes and the like. Coadministeringsuch agents can decrease the dosage of a therapeutic agent (e.g.,therapeutic antibody or antibody chain) needed to achieve a desiredeffect.

C. Pharmaceutical Compositions

Agents of the invention are often administered as pharmaceuticalcompositions comprising an active therapeutic agent, i.e., and a varietyof other pharmaceutically acceptable components. See Remington'sPharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pa.(1980)). The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions can alsoinclude, depending on the formulation desired,pharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenicstabilizers and the like.

Pharmaceutical compositions can also include large, slowly metabolizedmacromolecules such as proteins, polysaccharides such as chitosan,polylactic acids, polyglycolic acids and copolymers (such as latexfunctionalized Sepharose™, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (such as oildroplets or liposomes). Additionally, these carriers can function asimmunostimulating agents (i.e., adjuvants).

For parenteral administration, agents of the invention can beadministered as injectable dosages of a solution or suspension of thesubstance in a physiologically acceptable diluent with a pharmaceuticalcarrier that can be a sterile liquid such as water oils, saline,glycerol, or ethanol. Additionally, auxiliary substances, such aswetting or emulsifying agents, surfactants, pH buffering substances andthe like can be present in compositions. Other components ofpharmaceutical compositions are those of petroleum, animal, vegetable,or synthetic origin, for example, peanut oil, soybean oil, and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers, particularly for injectable solutions.Antibodies can be administered in the form of a depot injection orimplant preparation, which can be formulated in such a manner as topermit a sustained release of the active ingredient. An exemplarycomposition comprises monoclonal antibody at 5 mg/mL, formulated inaqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted topH 6.0 with HCl.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect, as discussed above (see Langer, Science 249:1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28:97 (1997)). Theagents of this invention can be administered in the form of a depotinjection or implant preparation, which can be formulated in such amanner as to permit a sustained or pulsatile release of the activeingredient.

Additional formulations suitable for other modes of administrationinclude oral, intranasal, and pulmonary formulations, suppositories, andtransdermal applications. For suppositories, binders and carriersinclude, for example, polyalkylene glycols or triglycerides; suchsuppositories can be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1%-2%. Oralformulations include excipients, such as pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, and magnesium carbonate. These compositions take the form ofsolutions, suspensions, tablets, pills, capsules, sustained releaseformulations or powders and contain 10%-95% of active ingredient,preferably 25%-70%.

Topical application can result in transdermal or intradermal delivery.Topical administration can be facilitated by co-administration of theagent with cholera toxin or detoxified derivatives or subunits thereofor other similar bacterial toxins (See Glenn et al., Nature 391, 851(1998)). Co-administration can be achieved by using the components as amixture or as linked molecules obtained by chemical crosslinking orexpression as a fusion protein.

Alternatively, transdermal delivery can be achieved using a skin path orusing transferosomes (Paul et al., Eur. J. Immunol. 25:3521 (1995); Cevcet al., Biochem. Biophys. Acta 1368:201-15 (1998)).

III. Monitoring the Course of Treatment

The invention provides methods of monitoring treatment in a patientsuffering from or susceptible to Alzheimer's, i.e., for monitoring acourse of treatment being administered to a patient. The methods can beused to monitor both therapeutic treatment on symptomatic patients andprophylactic treatment on asymptomatic patients. In particular, themethods are useful for monitoring passive immunization (e.g., measuringlevel of administered antibody).

Some methods entail determining a baseline value, for example, of anantibody level or profile in a patient, before administering a dosage ofagent, and comparing this with a value for the profile or level aftertreatment. A significant increase (i.e., greater than the typical marginof experimental error in repeat measurements of the same sample,expressed as one standard deviation from the mean of such measurements)in value of the level or profile signals a positive treatment outcome(i.e., that administration of the agent has achieved a desiredresponse). If the value for immune response does not changesignificantly, or decreases, a negative treatment outcome is indicated.

In other methods, a control value (i.e., a mean and standard deviation)of level or profile is determined for a control population. Typicallythe individuals in the control population have not received priortreatment. Measured values of the level or profile in a patient afteradministering a therapeutic agent are then compared with the controlvalue. A significant increase relative to the control value (e.g.,greater than one standard deviation from the mean) signals a positive orsufficient treatment outcome. A lack of significant increase or adecrease signals a negative or insufficient treatment outcome.Administration of agent is generally continued while the level isincreasing relative to the control value. As before, attainment of aplateau relative to control values is an indicator that theadministration of treatment can be discontinued or reduced in dosageand/or frequency.

In other methods, a control value of the level or profile (e.g., a meanand standard deviation) is determined from a control population ofindividuals who have undergone treatment with a therapeutic agent andwhose levels or profiles have plateaued in response to treatment.Measured values of levels or profiles in a patient are compared with thecontrol value. If the measured level in a patient is not significantlydifferent (e.g., more than one standard deviation) from the controlvalue, treatment can be discontinued. If the level in a patient issignificantly below the control value, continued administration of agentis warranted. If the level in the patient persists below the controlvalue, then a change in treatment may be indicated.

In other methods, a patient who is not presently receiving treatment buthas undergone a previous course of treatment is monitored for antibodylevels or profiles to determine whether a resumption of treatment isrequired. The measured level or profile in the patient can be comparedwith a value previously achieved in the patient after a previous courseof treatment. A significant decrease relative to the previousmeasurement (i.e., greater than a typical margin of error in repeatmeasurements of the same sample) is an indication that treatment can beresumed. Alternatively, the value measured in a patient can be comparedwith a control value (mean plus standard deviation) determined in apopulation of patients after undergoing a course of treatment.Alternatively, the measured value in a patient can be compared with acontrol value in populations of prophylactically treated patients whoremain free of symptoms of disease, or populations of therapeuticallytreated patients who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the control level(i.e., more than a standard deviation) is an indicator that treatmentshould be resumed in a patient.

The tissue sample for analysis is typically blood, plasma, serum, mucousfluid or cerebrospinal fluid from the patient. The sample is analyzed,for example, for levels or profiles of antibodies to Aβ peptide, e.g.,levels or profiles of humanized antibodies. ELISA methods of detectingantibodies specific to Aβ are described in the Examples section. In somemethods, the level or profile of an administered antibody is determinedusing a clearing assay, for example, in an in vitro phagocytosis assay,as described herein. In such methods, a tissue sample from a patientbeing tested is contacted with amyloid deposits (e.g., from a PDAPPmouse) and phagocytic cells bearing Fc receptors. Subsequent clearing ofthe amyloid deposit is then monitored. The existence and extent ofclearing response provides an indication of the existence and level ofantibodies effective to clear Aβ in the tissue sample of the patientunder test.

The antibody profile following passive immunization typically shows animmediate peak in antibody concentration followed by an exponentialdecay. Without a further dosage, the decay approaches pretreatmentlevels within a period of days to months depending on the half-life ofthe antibody administered.

In some methods, a baseline measurement of antibody to Aβ in the patientis made before administration, a second measurement is made soonthereafter to determine the peak antibody level, and one or more furthermeasurements are made at intervals to monitor decay of antibody levels.When the level of antibody has declined to baseline or a predeterminedpercentage of the peak less baseline (e.g., 50%, 25% or 10%),administration of a further dosage of antibody is administered. In somemethods, peak or subsequent measured levels less background are comparedwith reference levels previously determined to constitute a beneficialprophylactic or therapeutic treatment regime in other patients. If themeasured antibody level is significantly less than a reference level(e.g., less than the mean minus one standard deviation of the referencevalue in population of patients benefiting from treatment)administration of an additional dosage of antibody is indicated.

Additional methods include monitoring, over the course of treatment, anyart-recognized physiologic symptom (e.g., physical or mental symptom)routinely relied on by researchers or physicians to diagnose or monitoramyloidogenic diseases (e.g., Alzheimer's disease). For example, one canmonitor cognitive impairment. The latter is a symptom of Alzheimer'sdisease and Down's syndrome but can also occur without othercharacteristics of either of these diseases. For example, cognitiveimpairment can be monitored by determining a patient's score on theMini-Mental State Exam in accordance with convention throughout thecourse of treatment.

C. Kits

The invention further provides kits for performing the monitoringmethods described above. Typically, such kits contain an agent thatspecifically binds to antibodies to Aβ. The kit can also include alabel. For detection of antibodies to the label is typically in the formof labeled anti-idiotypic antibodies. For detection of antibodies, theagent can be supplied prebound to a solid phase, such as to the wells ofa microtiter dish. Kits also typically contain labeling providingdirections for use of the kit. The labeling may also include a chart orother correspondence regime correlating levels of measured label withlevels of antibodies to Aβ. The term labeling refers to any written orrecorded material that is attached to, or otherwise accompanies a kit atany time during its manufacture, transport, sale or use. For example,the term labeling encompasses advertising leaflets and brochures,packaging materials, instructions, audio or videocassettes, computerdiscs, as well as writing imprinted directly on kits.

The invention also provides diagnostic kits, for example, research,detection and/or diagnostic kits (e.g., for performing in vivo imaging).Such kits typically contain an antibody for binding to an epitope of Aβ,preferably within residues 1-10. Preferably, the antibody is labeled ora secondary labeling reagent is included in the kit. Preferably, the kitis labeled with instructions for performing the intended application,for example, for performing an in vivo imaging assay. Exemplaryantibodies are those described herein.

D. In Vivo Imaging

The invention provides methods of in vivo imaging amyloid deposits in apatient. Such methods are useful to diagnose or confirm diagnosis ofAlzheimer's disease, or susceptibility thereto. For example, the methodscan be used on a patient presenting with symptoms of dementia. If thepatient has abnormal amyloid deposits, then the patient is likelysuffering from Alzheimer's disease. The methods can also be used onasymptomatic patients. Presence of abnormal deposits of amyloidindicates susceptibility to future symptomatic disease. The methods arealso useful for monitoring disease progression and/or response totreatment in patients who have been previously diagnosed withAlzheimer's disease.

The methods work by administering a reagent, such as antibody that bindsto Aβ, to the patient and then detecting the agent after it has bound.Preferred antibodies bind to Aβ deposits in a patient without binding tofull length APP polypeptide. Antibodies binding to an epitope of Aβwithin amino acids 1-10 are particularly preferred. In some methods, theantibody binds to an epitope within amino acids 7-10 of A. Suchantibodies typically bind without inducing a substantial clearingresponse. In other methods, the antibody binds to an epitope withinamino acids 1-7 of Aβ. Such antibodies typically bind and induce aclearing response to Aβ. However, the clearing response can be avoidedby using antibody fragments lacking a full-length constant region, suchas Fabs. In some methods, the same antibody can serve as both atreatment and diagnostic reagent. In general, antibodies binding toepitopes C-terminal to residue 10 of Aβ do not show as strong a signalas antibodies binding to epitopes within residues 1-10, presumablybecause the C-terminal epitopes are inaccessible in amyloid deposits.Accordingly, such antibodies are less preferred.

Diagnostic reagents can be administered by intravenous injection intothe body of the patient, or directly into the brain by intracranialinjection or by drilling a hole through the skull. The dosage of reagentshould be within the same ranges as for treatment methods. Typically,the reagent is labeled, although in some methods, the primary reagentwith affinity for Aβ is unlabelled and a secondary labeling agent isused to bind to the primary reagent. The choice of label depends on themeans of detection. For example, a fluorescent label is suitable foroptical detection. Use of paramagnetic labels is suitable fortomographic detection without surgical intervention. Radioactive labelscan also be detected using PET or SPECT.

Diagnosis is performed by comparing the number, size, and/or intensityof labeled loci, to corresponding baseline values. The base line valuescan represent the mean levels in a population of undiseased individuals.Baseline values can also represent previous levels determined in thesame patient. For example, baseline values can be determined in apatient before beginning treatment, and measured values thereaftercompared with the baseline values. A decrease in values relative tobaseline signals a positive response to treatment.

The present invention will be more fully described by the followingnon-limiting examples.

EXAMPLES

The following Sequence identifiers are used throughout the Examplessection to refer to immunoglobulin chain variable region nucleotide andamino acid sequences.

VL nucleotide VL amino acid VH nucleotide VH amino acid Antibodysequence sequence sequence sequence 12B4 SEQ ID NO: 1 SEQ ID NO: 2 SEQID NO: 3 SEQ ID NO: 4 humanized SEQ ID NO: 5 SEQ ID NO: 6 SEQ ID NO: 7SEQ ID NO: 8 12B4v1 humanized SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 9 SEQID NO: 10 12B4v2 humanized SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 11 SEQID NO: 12 12B4v3 germline A19 SEQ ID NO: 29 SEQ ID NO: 30 Kabat ID SEQID NO: 31 SEQ ID NO: 32 005036 Kabat ID SEQ ID NO: 33 SEQ ID NO: 34000333 germline SEQ ID NO: 35 SEQ ID NO: 36 VH4-61 germline SEQ ID NO:37 SEQ ID NO: 38 VH4-39

As used herein, an antibody or immunoglobulin sequence comprising a VLand/or VH sequence as set forth in any one of SEQ ID NOs: 1-12 or 29-38can comprise either the full sequence or can comprise the maturesequence (i.e., mature peptide without the signal or leader peptide).

Example I Cloning and Sequencing of the Mouse 12B4 Variable Regions

Cloning and Sequence Analysis of 12B4 VH. The VH and VL regions of 12B4from hybridoma cells were cloned by RT-PCR and 5′ RACE using mRNA fromhybridoma cells and standard cloning methodology. The nucleotidesequence (SEQ ID NO:3) and deduced amino acid sequence (SEQ ID NO:4)derived from two independent cDNA clones encoding the presumed 12B4 VHdomain, are set forth in Table 2 and Table 3, respectively.

TABLE 2 Mouse 12B4 VH DNA sequence.ATGGACAGGCTTACTTCCTCATTCCTGCTGCTGATTGTCCCTGCATATGTCCTGTCCCAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTAATGGTATGGGTGTGAGCTGGATTCGTCAGCCTTCAGGAAAGGGTCTGGAGTGGCTGGCACACATTTACTGGGATGAGGACAAGCGCTATAACCCATCCCTGAAGAGCCGGCTCACAATCTCCAAGGATACCTCTAACAATCAGGTATTCCTCAAGATCACCAATGTGGACACTGCTGATACTGCCACATACTACTGTGCTCGAAGGAGGATCATCTATGATGTTGAGGACTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAG (SEQ ID NO: 3) *Leader peptideunderlined.

TABLE 3 Mouse 12B4 VH amino acid sequencemdrltssflllivpayvlsqVTLKESGPGILQPSQTLSLTCSFSGFSLStngmgvsWIRQPSGKGLEWLAhiywdedkrynpslksRLTISKDTSNNQVFLKITNVDTADTATYYCARrriiydvedyfdyWGQGTTLTVSS (SEQ ID NO: 4) *Leaderpeptide and CDRs in lower case.

Cloning and Sequence Analysis of 12B4 VL. The light chain variable VLregion of 12B4 was cloned in an analogous manner as the VH region. Thenucleotide sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQID NO:2) derived from two independent cDNA clones encoding the presumed12B4 VL domain, are set forth in Table 4 and Table 5, respectively.

TABLE 4 Mouse 12B4 VL DNA sequenceATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAACATTGTTCATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAA AC (SEQ ID NO: 1)*Leader peptide underlined

TABLE 5 Mouse 12B4 VL amino acid sequencemklpvrllvlmfwipasssDVLMTQTPLSLPVSLGDQASISCrssqnivhsngntyleWYLQKPGQSPKLLIYkvsnrfSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCfqgshvpltFGAGTKLELK (SEQ ID NO: 2) *Leader peptide andCDRs in lower case.

The 12B4 VL and VH sequences meet the criteria for functional V regionsin so far as they contain a contiguous ORF from the initiator methionineto the C-region, and share conserved residues characteristic ofimmunoglobulin V region genes. From N-terminal to C-terminal, both lightand heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is in accordancewith the numbering convention of Kabat et al., supra.

Example II Expression of Chimeric 12B4 Antibody

Expression of Chimeric 12B4 Antibody: The variable heavy and light chainregions were re-engineered to encode splice donor sequences downstreamof the respective VDJ or VJ junctions, and cloned into the mammalianexpression vector pCMV-hγ1 for the heavy chain, and pCMV-hκ1 for thelight chain. These vectors encode human γ1 and Ck constant regions asexonic fragments downstream of the inserted variable region cassette.Following sequence verification, the heavy chain and light chainexpression vectors were co-transfected into COS cells. Various heavychain clones were independently co-transfected with different chimericlight chain clones to confirm reproducibility of the result. Conditionedmedia was collected 48 hrs post transfection and assayed by western blotanalysis for antibody production or ELISA for Aβ binding. The multipletransfectants all expressed heavy chain+light chain combinations whichare recognized by a goat anti-human IgG (H+L) antibody on a westernblot.

Direct binding of chimeric 12B4 antibodies to Aβ was tested by ELISAassay. FIG. 5 demonstrates that chimeric 12B4 was found to bind to Aβwith high avidity, similar to that demonstrated by chimeric andhumanized 3D6 (FIG. 5). (The cloning, characterization and humanizationof 3D6 is described in U.S. patent application Ser. No. 10/010,942, theentire content of which is incorporated herein by this reference.)Furthermore, an ELISA based competitive inhibition assay revealed thatthe chimeric 12B4 and the murine 12B4 antibody competed equally withbiotinylated murine and chimeric 3D6, as well as 10D5 (a murinemonoclonal antibody of the IgGγ1 isotype which recognizes the sameepitope as 12B4), in binding to Aβ. FIG. 6 demonstrates that chimeric12B4 (dashed line, open triangles) competes with equal potency with itsnon biotinylated murine counterpart (solid line, closed triangles) forbinding of biotinylated murine 12B4 to Aβ1-42 peptide.

Example III Efficacy of mAb 12B4 on Various Neuropathological Endpointsin PDAPP Mice

This Example describes the efficacy of murine mAb 12B4 on variousneuropathological endpoints. Comparison of two mAbs, 12B4 and 3D6, isdescribed. Both mAbs are of the IgG2a isotype and both bind an epitopewithin the N-terminus of the Aβ peptide.

Immunizations

PDAPP mice were passively immunized with either mAb 12B4 (recognizingAβ3-7) or mAb 3D6 (recognizing Aβ1-5), both of the IgGγ2a isotype. 12B4was tested at 10 mg/kg. 3D6 was administered at three different doses,10 mg/kg, 1 mg/kg and 10 mg/kg once a month (1×4). An unrelated IgG2aantibody (TY 11/15) and PBS injections served as controls. Activeimmunization with Aβ peptide served as a comparison. Between 20 and 35animals were analyzed in each group.

The neuropathological endpoints assayed include amyloid burden andneuritic burden.

Amyloid Burden

The extent of the frontal cortex occupied by amyloid deposits wasdetermined by immunostaining with 3D6 followed by quantitative imageanalysis. The results of this analysis are shown in Table 6. All of theimmunotherapies (e.g., immunization with 12B4, 3D6 (all doses tested)and Aβ peptide) led to a significant reduction of amyloid burden.

Neuritic Burden

Previously, it had been observed that 10D5 was unable to significantlyreduce neuritic burden, suggesting that antibodies of the IgGγ2aisotype, but not other isotypes, are able to reduce neuritic burden inanimal models of Alzheimer's disease (data not shown). Neuritic burdenfollowing passive immunization with 12B4 versus 3D6 (both of the IgGγ2aisotype) was thus determined in PDAPP mice by immunostaining of brainsections with anti-APP antibody 8E5 followed by quantitative imageanalysis. Neuritic dystrophy is indicated by the appearance ofdystrophic neurites (e.g., neurites with a globular appearance) locatedin the immediate vicinity of amyloid plaques. The results of thisanalysis are shown in Table 7. These data indicate that treatment with12B4 most significantly reduced neuritic burden. By contrast, 3D6 didnot significantly reduce neuritic burden.

TABLE 6 Frontal Cortex Amyloid Burden TY 3D6, 3D6, 3D6, PBS 11/15 12B410 mg/kg 1 mg/kg 10 mg/kg/4 wks. Active N 31 30 33 29 31 32 24 Median (%AB) 15.182297 13.303288 2.317222 0.865671 2.286513 1.470956 2.162772Range 0.160-31.961 0-61.706 0-14.642 0-7.064 0.077-63.362 0-10.6880-30.715 p Value (*M-W) .9425 ns ***<.0001 ***.0001 ***<.0001 ***<.0001***.0004 % Change N/A 12% 85% 94% 85% 90% 86%

TABLE 7 Frontal Cortex Neuritic Burden TY 3D6, 3D6, 3D6, PBS 11/15 12B410 mg/kg 1 mg/kg 10 mg/kg/4 wks. Active N 31 30 33 29 31 32 24 Median (%NB) 0.3946 0.3958 0.0816 0.4681 0.3649 0.4228 0.2344 Range 0-1.38280-2.6800 0-0.8127 0-1.3098 0-1.5760 0-1.8215 0-1.1942 p Value (*M-W).8967 ns ***.0002 .9587 ns .6986 ns >.9999 ***.0381 % Change 0% 79% 0%7% 0% 41%

The above results indicate that treatment with an Aβ antibody of theIgGγ2a isotype may be necessary, but not sufficient, to reduce neuriticburden. Binding Aβ via a distinct epitope (i.e., Aβ3-7) may also beessential to reduce this pathology in PDAPP mice.

The characterization of various neuropathological endpoints in the PDAPPmouse model of Alzheimer's disease provides valuable information to beused by the skilled artisan in designing appropriate human therapeuticimmunization protocols. For example, reduction of neuritic burden may beaccomplished in human subjects using a humanized version of 12B4 of theIgG1 subtype (i.e., the human equivalent of the IgGγ2A subtype in mice)which binds to an epitope within residues 3-7 of Aβ.

Example IV Ex Vivo Screening Assay for Activity of Antibodies AgainstAmyloid Deposits

To examine the effect of antibodies on plaque clearance, an ex vivoassay was utilized in which primary microglial cells were cultured withunfixed cryostat sections of either PDAPP mouse or human AD brains.Microglial cells were obtained from the cerebral cortices of neonateDBA/2N mice (1-3 days). The cortices were mechanically dissociated inHBSS⁻⁻ (Hanks' Balanced Salt Solution, Sigma) with 50 μg/ml DNase I(Sigma). The dissociated cells were filtered with a 100 μm cell strainer(Falcon), and centrifuged at 1000 rpm for 5 minutes. The pellet wasresuspended in growth medium (high glucose DMEM, 10% FBS, 25 ng/mlrecombinant murine GM-CSF (rmGM-CSF), and the cells were plated at adensity of 2 brains per T-75 plastic culture flask. After 7-9 days, theflasks were rotated on an orbital shaker at 200 rpm for 2 h at 37° C.The cell suspension was centrifuged at 1000 rpm and resuspended in theassay medium.

10-μm cryostat sections of PDAPP mouse or human AD brains (post-morteminterval <3 hr) were thaw mounted onto poly-lysine coated round glasscoverslips and placed in wells of 24-well tissue culture plates. Thecoverslips were washed twice with assay medium consisting of H—SFM(Hybridoma-serum free medium, Gibco BRL) with 1% FBS, glutamine,penicillin/streptomycin, and 5 ng/ml rmGM-CSF (R&D). Control or anti-Aβantibodies (12B4) were added at a 2× concentration (5 μg/ml final) for 1hour. The microglial cells were then seeded at a density of 0.8×10⁶cells/ml assay medium. The cultures were maintained in a humidifiedincubator (37° C., 5% CO₂) for 24 hr or more. At the end of theincubation, the cultures were fixed with 4% paraformaldehyde andpermeabilized with 0.1% Triton-X100. The sections were stained withbiotinylated 3D6 followed by a streptavidin/Cy3 conjugate (JacksonImmunoResearch). The exogenous microglial cells were visualized by anuclear stain (DAPI). The cultures were observed with an invertedfluorescent microscope (Nikon, TE300) and photomicrographs were takenwith a SPOT digital camera using SPOT software (Diagnostic instruments).

When the assay was performed with PDAPP brain sections in the presenceof a control antibody having no in vivo efficacy, β-amyloid plaquesremained intact and no phagocytosis was observed. In contrast, whenadjacent sections were cultured in the presence of 3D6 or 12B4, theamyloid deposits were largely gone and the microglial cells showednumerous phagocytic vesicles containing Aβ (FIG. 7). Similar resultswere obtained with AD brain sections; 3D6 (a humanized version) andchimeric 12B4 induced phagocytosis of AD plaques, while control IgG1 wasineffective (FIG. 8A-B).

The data presented in Examples II and III confirm function of the cloned12B4 variable regions.

Example V 12B4 Humanization

A. 12B4 Humanized Antibody, Version 1

Homology/Molecular Modeling. In order to identify key structuralframework residues in the murine 12B4 antibody, a three-dimensionalmodel was generated based on the closest murine antibodies for the heavyand light chains. For this purpose, an antibody designated 2PCP waschosen as a template for modeling the 12B4 light chain (PDB ID: 2PCP,Lim et al. (1998) J. Biol. Chem. 273:28576), and an antibody designated1ETZ was chosen as the template for modeling the heavy chain. (PDB ID:1ETZ, Guddat et al. (2000) J. Mol. Biol. 302:853). Amino acid sequencealignment of 12B4 with the light chain and heavy chain of theseantibodies revealed that the 2PCP and 1ETZ antibodies share significantsequence homology with 12B4. In addition, the CDR loops of the selectedantibodies fall into the same canonical Chothia structural classes as dothe CDR loops of 12B4. Therefore, 2PCP and 1ETZ were initially selectedas antibodies of solved structure for homology modeling of 12B4.

A first pass homology model of 12B4 variable region based on theantibodies noted above was constructed using the Look & SegMod ModulesGeneMine (v3.5) software package. This software was purchased under aperpetual license from Molecular Applications Group (Palo Alto, Calif.).This software package, authored by Drs. Michael Levitt and Chris Lee,facilitates the process of molecular modeling by automating the stepsinvolved in structural modeling a primary sequence on a template ofknown structure based on sequence homology. Working on a SiliconGraphics IRIS workstation under a UNIX environment, the modeledstructure is automatically refined by a series of energy minimizationsteps to relieve unfavorable atomic contacts and optimize electrostaticand van der Walls interactions. A further refined model was built usingthe modeling capability of Quanta®.

Selection of Human Acceptor Antibody Sequences. Suitable human acceptorantibody sequences were identified by computer comparisons of the aminoacid sequences of the mouse variable regions with the sequences of knownhuman antibodies. The comparison was performed separately for the 12B4heavy and light chains. In particular, variable domains from humanantibodies whose framework sequences exhibited a high degree of sequenceidentity with the murine VL and VH framework regions were identified byquery of the Kabat Database using NCBI BLAST (publicly accessiblethrough the National Institutes of Health NCBI internet server) with therespective murine framework sequences.

Two candidate sequences were chosen as acceptor sequences based on thefollowing criteria: (1) homology with the subject sequence; (2) sharingcanonical CDR structures with the donor sequence; and (3) not containingany rare amino acid residues in the framework regions. The selectedacceptor sequence for VL is Kabat ID Number (KABID) 005036 (GenbankAccession No. X67904), and for VH is KABID 000333 (Genbank Accession No.X54437). First versions of humanized 3D6 antibody utilize these selectedacceptor antibody sequences.

Substitution of Amino Acid Residues. As noted supra, the humanizedantibodies of the invention comprise variable framework regionssubstantially from a human immunoglobulin (acceptor immunoglobulin) andcomplementarity determining regions substantially from a mouseimmunoglobulin (donor immunoglobulin) termed 12B4. Having identified thecomplementarity determining regions of 12B4 and appropriate humanacceptor immunoglobulins, the next step was to determine which, if any,residues from these components to substitute to optimize the propertiesof the resulting humanized antibody.

Reshaped Light Chain V Region:

The amino acid alignment of the reshaped light chain V region is shownin FIG. 1. The choice of the acceptor framework (Kabid 005036) is fromthe same human subgroup as that which corresponds to the murine Vregion, has no unusual framework residues, and the CDRs belong to thesame Chothia canonical structure groups. A single back mutation (12V) isdictated as this residue falls into the canonical classification.Version 1 of the reshaped VL is fully germline.

Reshaped Heavy Chain V Region:

The amino acid alignment of the reshaped heavy chain V region is shownin FIG. 2. The choice for the acceptor framework (Kabid 000333) is fromthe same human subgroup as that which corresponds to the murine Vregion, has no unusual framework residues, and the CDRs belong to thesame Chothia canonical groups. Structural modeling of the murine VHchain, in conjunction with the amino acid alignment of Kabid 000333 tothe murine sequence dictates 9 back-mutations in version 1 (v1) of thereshaped heavy chain: L2V, V24F, G27F, 129L, 148L, G49A, V67L, V71K, &F78V (Kabat numbering). The back mutations are highlighted by asterisksin the amino-acid alignment shown in FIG. 2.

Of the 9 back mutations, 4 are dictated by the model because theresidues are canonical residues (V24F, G27F, 129L, & V71K, indicated bysolid filled boxes), i.e. framework residues which may contribute toantigen binding by virtue of proximity to CDR residues. There are noback mutations necessary in the next most important class of residues,the interface residues involved in VH-VL packing interactions (indicatedby open boxes). The remaining 5 residues targeted for back mutation(L2V, 148L, G49A, V67L, F78V, Kabat numbering) all fall into the vernierclass (indirect contribution to CDR conformation, dense stippled boxesin FIG. 2,).

Version 2 was designed to retain the lowest number of non-CDR murineresidues. The L2V backmutation introduces a non-germline change (whenusing VH4-61 as the germline reference), and this backmutation iseliminated in version 2 of the heavy chain to restore it to germ line.The remaining 4 vernier class back mutations are also restored inversion 2 of the heavy chain (148L, G49A, V67L, F78V). Version 2 thuscontain a total of 5 non-CDR murine residues (1 in VL, and 4 in VH).Version 3 was designed to restore 2 of the 5 vernier residues (148L, &F78V), which the model indicates may be the more important vernierresidues. Hence version 3 contains a total of 7 non CDR murine residues.

A summary of the changes incorporated into versions 1, 2 and 3 ofhumanized 12B4 are presented in Table 8.

TABLE 8 Summary of changes in humanized 12B4.v1 Changes VL (111residues) VH (123 residues) Hu −> Mu: 1/111 9/123 Framework CDR1 8/167/7 CDR2 3/7 8/16 CDR3 6/8 10/13 Total Hu −> Mu 18/111 (16%) 34/123(28%, v2 = 23%) Mu −> Hu: 10/111 16/123 Framework Backmutation 1. I2V: acanonical 2. Canonical: V24F, notes position. G27F, I29L, & V71K 3.Packing: none. 4. Vernier: L2V*, I48L*#, G49A*, V67L*, F78V*# Acceptornotes 5. KABID 005036/ 8. KABID000333/Genbank Genbank Acc#-x67904Acc#x54437 6. CDRs from same 9. CDRs from same canonical structuralcanonical structural group group as donor mouse; as donor mouse; 7.anti-cardiolipin/ss DNA 10. rheumatoid factor mAb autoantibody from SLEfrom RA patient patient; *eliminate in v2 and v3; #eliminate in v2,restore in v3.

Tables 10 and 11 set forth Kabat numbering keys for the various lightand heavy chains, respectively.

TABLE 9 Key to Kabat Numbering for Light Chain mouse HUM A19- KAB 12B412B4 KABID Germ- # # TYPE VL VL 005036 line Comment  1 1 FR1 D D D D  22 V V I I canonical - backmutate in v1, v2 and v3  3 3 L V V V  4 4 M MM M  5 5 T T T T  6 6 Q Q Q Q  7 7 T S S S  8 8 P P P P  9 9 L L L L 1010 S S S S 11 11 L L L L 12 12 P P P P 13 13 V V V V 14 14 S T T T 15 15L P P P 16 16 G G G G 17 17 D E E E 18 18 Q P P P 19 19 A A A A 20 20 SS S S 21 21 I I I I 22 22 S S S S 23 23 C C C C 24 24 CDR1 R R R R 25 25S S S S 26 26 S S S S 27 27 Q Q Q Q  27A 28 N N S S   27B 29 I I L L  27C 30 V V L L  27D 31 H H H H   27E 32 S S R S 28 33 N N Y N 29 34 GG G G 30 35 N N Y Y 31 36 T T N N 32 37 Y Y Y Y 33 38 L L L L 34 39 E ED D 35 40 FR2 W W W W 36 41 Y Y Y Y 37 42 L L L L 38 43 Q Q Q Q 39 44 KK K K 40 45 P P P P 41 46 G G G G 42 47 Q Q Q Q 43 48 S S S S 44 49 P PP P 45 50 K Q Q Q 46 51 L L L L 47 52 L L L L 48 53 I I I I 49 54 Y Y YY 50 55 CDR2 K K L L 51 56 V V G G 52 57 S S S S 53 58 N N N N 54 59 R RR R 55 60 F F A A 56 61 S S S S 57 62 FR3 G G G G 58 63 V V V V 59 64 PP P P 60 65 D D D D 61 66 R R R R 62 67 F F F F 63 68 S S S S 64 69 G GG G 65 70 S S S S 66 71 G G G G 67 72 S S S S 68 73 G G G G 69 74 T T TT 70 75 D D D D 71 76 F F F F 72 77 T T T T 73 78 L L L L 74 79 K K K K75 80 I I I I 76 81 S S S S 77 82 R R R R 78 83 V V V V 79 84 E E E E 8085 A A A A 81 86 E E E E 82 87 D D D D 83 88 L V V V 84 89 G G G G 85 90V V V V 86 91 Y Y Y Y 87 92 Y Y Y Y 88 93 C C C C 89 94 CDR3 F F M M 9095 Q Q Q Q 91 96 G G A A 92 97 S S L L 93 98 H H Q Q 94 99 V V T T 95100 P P P P 96 101 L L Y 97 102 T T T 98 103 FR4 F F F 99 104 G G G 100 105 A Q Q 101  106 G G G 102  107 T T T 103  108 K K K 104  109 L L L105  110 E E E 106  111 L I I  106A 112 K K K

TABLE 10 Key to Kabat Numbering for Heavy Chain Mouse HUM VH4-39 VH4-61KAB 12B4 12B4 KABID Germ- Germ- # # TYPE VH VHv1 000333 line lineComment  1 1 FR1 Q Q Q Q Q  2 2 V V L L V vernier - backmutate in v1only  3 3 T Q Q Q Q  4 4 L L L L L  5 5 K Q Q Q Q  6 6 E E E E E  7 7 SS S S S  8 8 G G G G G  9 9 P P P P P 10 10 G G G G G 11 11 I L L L L 1212 L V V V V 13 13 Q K K K K 14 14 P P P P P 15 15 S S S S S 16 16 Q E EE E 17 17 T T T T T 18 18 L L L L L 19 19 S S S S S 20 20 L L L L L 2121 T T T T T 22 22 C C C C C 23 23 S T T T T 24 24 F F V V V canonical -backmutate in v1, v2 and v3 25 25 S S S S S 26 26 G G G G G 27 27 F F GG G canonical - backmutate in v1, v2 and v3 28 28 S S S S S 29 29 L L II V canonical - backmutate in v1, v2 and v3 30 30 S S S S S 31 31 CDR1 TT R S S 32 32 N N G S G 33 33 G G S S G 34 34 M M H Y Y 35 35 G G Y Y Y 35A 36 V V W W W   35B 37 S S G G S 36 38 FR2 W W W W W 37 39 I I I I I38 40 R R R R R 39 41 Q Q Q Q Q 40 42 P P P P P 41 43 S P P P P 42 44 GG G G G 43 45 K K K K K 44 46 G G G G G 45 47 L L L L L 46 48 E E E E E47 49 W W W W W 48 50 L L I I I vernier - backmutate in v1 and v3 only49 51 A A G G G vernier - backmutate in v1 only 50 52 CDR2 H H S S Y 5153 I I I I I 52 54 Y Y Y Y Y 53 55 W W Y Y Y 54 56 D D S S S 55 57 E E GG G 56 58 D D N S S 57 59 K K T T T 58 60 R R Y Y N 59 61 Y Y F Y Y 6062 N N N N N 61 63 P P P P P 62 64 S S S S S 63 65 L L L L L 64 66 K K KK K 65 67 S S S S S 66 68 FR3 R R R R R 67 69 L L V V V vernier -backmutate in v1 only 68 70 T T T T T 69 71 I I I I I 70 72 S S S S S 7173 K K V V V canonical - backmutate in v1, v2 and v3 72 74 D D D D D 7375 T T T T T 74 76 S S S S S 75 77 N K K K K 76 78 N N N N N 77 79 Q Q QQ Q 78 80 V V F F F vernier - backmutate in v1 and v3 79 81 F S S S S 8082 L L L L L 81 83 K K K K K 82 84 I L L L L  82A 85 T S S S S   82B 86N S S S S   82C 87 V V V V V 83 88 D T T T T 84 89 T A A A A 85 90 A A AA A 86 91 D D D D D 87 92 T T T T T 88 93 A A A A A 89 94 T V V V V 9095 Y Y Y Y Y 91 96 Y Y Y Y Y 92 96 C C C C C 93 97 A A A A A 94 98 R R RR R 95 99 CDR3 R R L  95A 100 — — G 96 101 R R P 97 102 I I D 98 103 I ID 99 104 Y Y Y 100  105 D D T  100A 106 V V L  100B 107 E E D  100C 108D D G  100D 109 Y Y —  100E 110 F F M 101  111 D D D 102  112 Y Y V 103 113 FR4 W W W 104  114 G G G 105  115 Q Q Q 106  116 G G G 107  117 T TT 108  118 T T T 109  119 L V V 110  120 T T T 111  121 V V V 112  122 SS S 113  123 S S S

The humanized antibodies preferably exhibit a specific binding affinityfor Aβ of at least 10⁷, 10⁸, 10⁹ or 10¹⁰ M⁻¹. Usually the upper limit ofbinding affinity of the humanized antibodies for Aβ is within a factorof three, four or five of that of 12B4 (i.e., ˜10⁹ M⁻¹). Often the lowerlimit of binding affinity is also within a factor of three, four or fiveof that of 12B4.

Assembly and Expression of Humanized 12B4 VH and VL, Version 1 FIG. 9 ais a schematic representation of the strategy for PCR mediated assemblyof humanized VL.v1. FIG. 9 b is a schematic representation of thestrategy for PCR mediated assembly of humanized VH.v1. Table 11 setsforth primers used for the PCR-mediated assembly of 12B4v1.

TABLE 11 Synthetic oligonucleotides used in PCR mediated assembly of humanized 12B4 V regions, v1 DNA Coding # Sizestrand? Sequence Comments 4881 135 Yes gagattaagcttgccgccaccATGAGGCThum12B4 VLv1A primer CCCTGCTCAGCTCCTGGGGCTGCTAATGCsynthesized by Oligos etc. TCTGGGTCTCTGGATCCAGTGGGGATGTT nts 1 >115 12B4VLv1.cons, sense GTGATGACTCAGTCTCCACTCTCCCTGCCstrand. Adds HindIII site + CGTCACCCCTGGAGAGCCG Kozak consensus.SEQ ID NO: 13 4882 131 No AGGAGCTGTGGAGACTGCCCTGGCTTCTGhum12B4 VLv1B primer CAGGTACCATTCCAAATAGGTGTTTCCATsynthesized by Oligos etc. TACTATGAACAATGTTCTGACTAGACCTGReverse Complement DNA CAGGAGATGGAGGCCGGCTCTCCAGGGGTSequence 12B4VLv1.cons(85,215) GACGGGCAGGGAGAG SEQ ID NO:14 4883 132 YesTGCAGAAGCCAGGGCAGTCTCCACAGCTC hum12B4 VLv1C primerCTGATCTACAAAGTTTCCAACCGATTTTC synthesized by Oligos etc.TGGGGTCCCTGACAGGTTCAGTGGCAGTG nts 185 > 316 12B4VLv1.cons,GATCAGGCACAGATTTTACACTGAAAATC sense strand. AGCAGAGTGGAGGCTGSEQ ID NO: 15 4884 130 No tgatatggatccactcacGTTTGATCTCChum12B4 VLv1D primer AGCTTGGTCCCCTGACCGAACGTGAGCGGsynthesized by Oligos etc. AACATGTGAACCTTGAAAGCAGTAATAAAReverse Complement DNA CCCCAACATCCTCAGCCTCCACTCTGCTG SequenceATTTTCAGTGTAAA 12B4VLv1.cons(286,397) adds SEQ ID NO: 16 splice donor +BamHI site. 4885 143 Yes gagataaagcttgccgccaccATGAAGCAhum12B4 VHv1A primer CCTGTGGTTCTTCCTCCTGCTGGTGGCAGsynthesized by Oligos etc. CTCCCAGATGGGTCCTGTCCCAGGTGCAG12B4VHv1.cons nts 1-122, adds CTGCAGGAGTCGGGCCCAGGACTGGTGAAKozak consensus, and HindIII GCCTTCGGAGACCCTGTCCCTCACCTG siteSEQ ID NO: 17 4886 137 No TCCTCATCCCAATAGATGTGTGCCAGCCAhum12B4 VHv1B primer CTCCAGTCCCTTCCCTGGGGGCTGCCGGAsynthesized by Oligos etc. TCCAGCTCACACCCATACCATTAGTGCTCReverse Complement DNA AGGGAAAAACCAGAGAAAGTGCAGGTGAGSequence 12B4VHv1.cons(94,230) GGACAGGGTCTCCGAAGGCTT SEQ ID NO:18 4887138 Yes GTGGCTGGCACACATCTATTGGGATGAGG hum12B4 VHv1C primerACAAGCGCTATAACCCATCCCTCAAGAGT synthesized by Oligos etc.CGACTCACCATATCAAAGGACACGTCCAA 12B4VHv1.cons nts201-338GAACCAGGTATCCCTGAAGCTGAGCTCTG TGACCGCTGCAGACACGGCCGT SEQ ID NO:19 4888139 No tcatatggatccactcacCTGAGGAGACG hum12B4 VHv1D primerGTGACCGTGGTCCCTTGGCCCCAGTAGTC synthesized by Oligos etc.AAAGTAGTCCTCAACATCATAGATGATCC Reverse Complement DNATCCTTCTCGCACAGTAATACACGGCCGTG Sequence TCTGCAGCGGTCACAGAGCTCAG12B4VHv1.cons(307,427) adds SEQ ID NO: 20 splice donor + BamHI site to3′ end. 4889 22 Yes gag att aag ctt gcc gcc acc A hum 12B4 VLv1, A +B For SEQ ID NO: 21 primer % A + T = 45.45 [10]; % C + G = 54.55 [12]Davis, Bostein, Roth Melting Temp C. 64.54 4890 21 NoAGG AGC TGT GGA GAC TGC CCT hum 12B4 VLv1, A + B bak SEQ ID NO: 22primer % A + T = 38.10 [8]; % C + G = 61.90 [13] Davis, Botstein, RothMelting Temp C. 66.47 4891 20 Yes TGC AGA AGC CAG GGC AGT CThum 12B4 VLv1, C + D For primer SEQ ID NO: 23 % A + T = 40.00 [8]; % C +G = 60.00 [12] Davis, Botstein, Roth Melting Temp C. 64.50 4892 28 Notga tat gga tcc act cac GTT hum 12B4 VLv1, C + D bak primer TGA TCT C% A + T = 57.14 [16]; SEQ ID NO: 24 % C + G = 42.86 [12]Davis, Botstein, Roth Melting Temp C. 64.61 4893 23 Yesgag ata aag ctt gcc gcc acc hum 12B4 VHv1, A + B For AT primer % A + T =47.83 SEQ ID NO: 25 [11]; % C + G = 52.17 [12] Davis, Botstein, RothMelting Temp C. 64.55 4894 24 No TCC TCA TCC CAA TAG ATG TGThum 12B4 VHv1, A + B bak primer GCC % A + T = 50.00 [12]; SEQ ID NO: 26% C + G = 50.00 [12] Davis, Botstein, Roth Melting Temp C. 64.57 4895 22Yes GTG GCT GGC ACA CAT CTA TTG G hum 12B4 VHv1, C + D For primerSEQ ID NO: 27 % A + T = 45.45 [10]; % C + G = 54.55 [12]Davis, Botstein, Roth Melting Temp C. 64.54 4896 24 Notca tat gga tcc act cac CTG hum 12B4 VHv1, C + D For primer AGG % A +T = 50.00 [12]; SEQ ID NO: 28 % C + G = 50.00 [12]Davis, Botstein, Roth Melting Temp C. 64.57

Equimolar rations of VHv1A+VHv1B and VHv1C+VHv1D synthetic fragmentswere annealed as pairs, in separate reaction tubes using standardprocedures. The A+B annealing reaction was assembled using PCR withprimers A+B for and A+B back at 60° C. annealing, 25 cycles (for=forward and back=backwards, alternatively, rev or reverse). Likewisethe C+D annealing reaction was assembled using PCR primers C+Dfor andC+Dback under identical conditions. The PCR-assembled 5′ A+B half, and3′ C+D half, were gel purified for a final PCR-mediated assembly. Full Vregion assembly was effected by mixing the A+B assembled 5′ half withthe C+D 3′ half of the V-region, annealing, and extending by PCR usingVHv1A+B for primer, and VHv1C+D back primer. The full length VH and VLregions assembled in this manner were gel purified, and cloned intopCRScript for DNA sequence verification.

The nucleotide sequences of humanized 12B4VL (version 1) (SEQ ID NO:5)and 12B4VH (version 1) (SEQ ID NO: 7) are listed below as Tables 12 and13, respectively.

TABLE 12 Nucleotide sequence of humanized 12B4VLv1.ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTCTGGGTCTCTGGATCCAGTGGGGATGTTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAACATTGTTCATAGTAATGGAAACACCTATTTGGAATGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCTTTCAAGGTTCACATGTTCCGCTCACGTTCGGTCAGGGGACCAAGCTGGAGAT CAAAC

TABLE 13 Nucleotide sequence of humanized 12B4VHv1ATGAAGCACCTGTGGTTCTTCCTCCTGCTGGTGGCAGCTCCCAGATGGGTCCTGTCCCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTTTCTCTGGTTTTTCCCTGAGCACTAATGGTATGGGTGTGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGCTGGCACACATCTATTGGGATGAGGACAAGCGCTATAACCCATCCCTCAAGAGTCGACTCACCATATCAAAGGACACGTCCAAGAACCAGGTATCCCTGAAGCTGAGCTCTGTGACCGCTGCAGACACGGCCGTGTATTACTGTGCGAGAAGGAGGATCATCTATGATGTTGAGGACTACTTTGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG

B. Humanized 12B4 Version 2 Antibody

A second version of humanized 12B4 was created having each of thesubstitutions indicated for version 1, except for the L→V substitutionat residue 2, the I→L substitution at residue 48, the G→A substitutionat residue 49, the V→L substitution at residue 67 and the F→Vsubstitution at residue 78. The nucleotide sequences of humanized 3D6version 2 light and heavy chains are set forth as SEQ ID NOs: 9 and 11,respectively. The nucleotide sequences of humanized 3D6 version 2 lightand heavy chains are set forth as SEQ ID NOs: 1 and 9, respectively. Theamino acid sequences of humanized 3D6 version 2 light and heavy chainsare set forth as SEQ ID NOs: 2 and 10, respectively.

C. Humanized 12B4 Version 3 Antibody

A third version of humanized 12B4 was created having each of thesubstitutions indicated for version 1, except for the L→V substitutionat residue 2, the G→A substitution at residue 49 and the V→Lsubstitution at residue 67. The nucleotide sequences of humanized 3D6version 2 light and heavy chains are set forth as SEQ ID NOs: 1 and 9,respectively. The nucleotide sequences of humanized 3D6 version 3 lightand heavy chains are set forth as SEQ ID NOs: 1 and 11, respectively.The amino acid sequences of humanized 3D6 version 3 light and heavychains are set forth as SEQ ID NOs: 2 and 12, respectively.

Example VI Prevention and Treatment of Human Subjects

A single-dose phase I trial is performed to determine safety in humans.A therapeutic agent is administered in increasing dosages to differentpatients starting from about 0.01 the level of presumed efficacy, andincreasing by a factor of three until a level of about 10 times theeffective mouse dosage is reached.

A phase II trial is performed to determine therapeutic efficacy.Patients with early to mid Alzheimer's Disease defined using Alzheimer'sdisease and Related Disorders Association (ADRDA) criteria for probableAD are selected. Suitable patients score in the 12-26 range on theMini-Mental State Exam (MMSE). Other selection criteria are thatpatients are likely to survive the duration of the study and lackcomplicating issues such as use of concomitant medications that mayinterfere. Baseline evaluations of patient function are made usingclassic psychometric measures, such as the MMSE, and the ADAS, which isa comprehensive scale for evaluating patients with Alzheimer's Diseasestatus and function. These psychometric scales provide a measure ofprogression of the Alzheimer's condition. Suitable qualitative lifescales can also be used to monitor treatment. Disease progression canalso be monitored by MRI. Blood profiles of patients can also bemonitored including assays of immunogen-specific antibodies and T-cellsresponses.

Following baseline measurements, patients begin receiving treatment.They are randomized and treated with either therapeutic agent or placeboin a blinded fashion. Patients are monitored at least every six months.Efficacy is determined by a significant reduction in progression of atreatment group relative to a placebo group.

A second phase II trial is performed to evaluate conversion of patientsfrom non-Alzheimer's Disease early memory loss, sometimes referred to asage-associated memory impairment (AAMI) or mild cognitive impairment(MCI), to probable Alzheimer's disease as defined as by ADRDA criteria.Patients with high risk for conversion to Alzheimer's Disease areselected from a non-clinical population by screening referencepopulations for early signs of memory loss or other difficultiesassociated with pre-Alzheimer's symptomatology, a family history ofAlzheimer's Disease, genetic risk factors, age, sex, and other featuresfound to predict high-risk for Alzheimer's Disease. Baseline scores onsuitable metrics including the MMSE and the ADAS together with othermetrics designed to evaluate a more normal population are collected.These patient populations are divided into suitable groups with placebocomparison against dosing alternatives with the agent. These patientpopulations are followed at intervals of about six months, and theendpoint for each patient is whether or not he or she converts toprobable Alzheimer's Disease as defined by ADRDA criteria at the end ofthe observation.

Although the foregoing invention has been described in detail forpurposes of clarity of understanding, it will be obvious that certainmodifications may be practiced within the scope of the appended claims.All publications and patent documents cited herein, as well as textappearing in the figures and sequence listing, are hereby incorporatedby reference in their entirety for all purposes to the same extent as ifeach were so individually denoted.

From the foregoing it will be apparent that the invention provides for anumber of uses. For example, the invention provides for the use of anyof the antibodies to Aβ described above in the treatment, prophylaxis ordiagnosis of amyloidogenic disease, or in the manufacture of amedicament or diagnostic composition for use in the same.

We claim:
 1. A method for reducing neuritic burden in a subject in needthereof comprising administering to the subject an effective dose of ahumanized immunoglobulin which binds to an epitope within amino acids3-7 of beta amyloid peptide (Aβ), wherein the immunoglobulin is of theIgG1 isotype, such that neuritic burden is reduced, wherein thehumanized immunoglobulin comprises: (i) a light chain comprising thethree complementarity determining regions (CDRs) from the 12B4immunoglobulin light chain variable region sequence set forth as SEQ IDNO:2, and a variable framework region from a human acceptorimmunoglobulin light chain; and (ii) a heavy chain comprising the threecomplementarity determining regions (CDRs) from the 12B4 immunoglobulinheavy chain variable region sequence set forth as SEQ ID NO:4, and avariable framework region from a human acceptor immunoglobulin heavychain, provided that at least one framework residue in the light orheavy chain is substituted with the corresponding amino acid residuefrom the mouse 12B4 light or heavy chain variable region sequence,wherein the framework residue is selected from the group consisting of:(a) a residue that non-covalently binds antigen directly; (b) a residueadjacent to a CDR; (c) a CDR-interacting residue; and (d) a residueparticipating in the VL-VH interface.
 2. The method of claim 1, whereinthe subject has an amyloidogenic disease.
 3. The method of claim 2,wherein the amyloidogenic disease is Alzheimer's disease.
 4. A methodfor treating an amyloidogenic disease in a patient comprisingadministering to the patient an effective dose of a humanizedimmunoglobulin capable of reducing beta amyloid peptide (Aβ) burden andneuritic dystrophy in the patient, wherein the immunoglobulin binds toan epitope within amino acids 3-7 of Aβ and is of the IgG1 isotype, suchthat a beneficial therapeutic response in said patient is generated,wherein the humanized immunoglobulin comprises: (i) a light chaincomprising the three complementarity determining regions (CDRs) from the12B4 immunoglobulin light chain variable region sequence set forth asSEQ ID NO:2, and a variable framework region from a human acceptorimmunoglobulin light chain; and (ii) a heavy chain comprising the threecomplementarity determining regions (CDRs) from the 12B4 immunoglobulinheavy chain variable region sequence set forth as SEQ ID NO:4, and avariable framework region from a human acceptor immunoglobulin heavychain, provided that at least one framework residue in the light orheavy chain is substituted with the corresponding amino acid residuefrom the mouse 12B4 light or heavy chain variable region sequence,wherein the framework residue is selected from the group consisting of:(a) a residue that non-covalently binds antigen directly; (b) a residueadjacent to a CDR; (c) a CDR-interacting residue; and (d) a residueparticipating in the VL-VH interface.
 5. The method of claim 4, whereinthe amyloidogenic disease is Alzheimer's disease.
 6. A method forreducing beta amyloid peptide (Aβ) burden and neuritic dystrophy in amammal comprising administering to the mammal an effective dose of ahumanized immunoglobulin capable of reducing beta amyloid peptide (Aβ)burden and neuritic dystrophy, wherein the immunoglobulin binds to anepitope within amino acids 3-7 of Aβ and is of the IgG1 isotype, suchthat Aβ burden and neuritic dystrophy are reduced, wherein the humanizedimmunoglobulin comprises: (i) a light chain comprising the threecomplementarity determining regions (CDRs) from the 12B4 immunoglobulinlight chain variable region sequence set forth as SEQ ID NO:2, and avariable framework region from a human acceptor immunoglobulin lightchain; and (ii) a heavy chain comprising the three complementaritydetermining regions (CDRs) from the 12B4 immunoglobulin heavy chainvariable region sequence set forth as SEQ ID NO:4, and a variableframework region from a human acceptor immunoglobulin heavy chain,provided that at least one framework residue in the light or heavy chainis substituted with the corresponding amino acid residue from the mouse12B4 light or heavy chain variable region sequence, wherein theframework residue is selected from the group consisting of: (a) aresidue that non-covalently binds antigen directly; (b) a residueadjacent to a CDR; (c) a CDR-interacting residue; and (d) a residueparticipating in the VL-VH interface.
 7. The method of claim 6, whereinthe mammal is a human.
 8. The method of claim 6 or 7, wherein the mammalhas an amyloidogenic disease.
 9. The method of claim 8, wherein theamyloidogenic disease is Alzheimer's disease.
 10. A method for treatingan amyloidogenic disease in a patient comprising administering to thepatient an effective dose of a humanized immunoglobulin which binds tosoluble beta amyloid peptide (Aβ) and reduces neuritic dystrophy in thepatient, wherein the immunoglobulin binds to an epitope within aminoacids 3-7 of Aβ and is of the IgG1 isotype, such that a beneficialtherapeutic response in said patient is generated, wherein the humanizedimmunoglobulin comprises: (i) a light chain comprising the threecomplementarity determining regions (CDRs) from the 12B4 immunoglobulinlight chain variable region sequence set forth as SEQ ID NO:2, and avariable framework region from a human acceptor immunoglobulin lightchain; and (ii) a heavy chain comprising the three complementaritydetermining regions (CDRs) from the 12B4 immunoglobulin heavy chainvariable region sequence set forth as SEQ ID NO:4, and a variableframework region from a human acceptor immunoglobulin heavy chain,provided that at least one framework residue in the light or heavy chainis substituted with the corresponding amino acid residue from the mouse12B4 light or heavy chain variable region sequence, wherein theframework residue is selected from the group consisting of: (a) aresidue that non-covalently binds antigen directly; (b) a residueadjacent to a CDR; (c) a CDR-interacting residue; and (d) a residueparticipating in the VL-VH interface.
 11. The method of claim 10,wherein the amyloidogenic disease is Alzheimer's disease.
 12. The methodof any one of claims 1, 4, 6 and 10, wherein the framework residue inthe light or heavy chain is selected from the group consisting of aresidue capable of interacting with antigen, a residue proximal to theantigen binding site, a residue capable of interacting with a CDR, aresidue adjacent to a CDR, a residue within 6 Å of a CDR residue, acanonical residue, a vernier zone residue, an interchain packingresidue, a rare residue, and a glycosylation site residue on the surfaceof the three-dimensional model.
 13. The method of any one of claims 1,4, 6 and 10, wherein the humanized immunoglobulin comprises threecomplementarity determining regions (CDRs) from the monoclonal antibody12B4 light chain variable region sequence set forth as SEQ ID NO:2 andvariable region framework residue L2 (Kabat numbering convention) fromthe monoclonal antibody 12B4 light chain, wherein the remainder of thelight chain is from a human immunoglobulin, and a heavy chain selectedfrom the group consisting of: (i) a heavy chain comprising threecomplementarity determining regions (CDRs) from the monoclonal antibody12B4 heavy chain variable region sequence set forth as SEQ ID NO:4 andvariable framework residues H2, H24, H27, H29, H48, H49, H67, H71, andH78 (Kabat numbering convention) from the monoclonal antibody 12B4 heavychain variable region sequence set forth as SEQ ID NO:4, wherein theremainder of the heavy chain is from a human immunoglobulin, (ii) aheavy chain comprising three complementarity determining regions (CDRs)from the monoclonal antibody 12B4 heavy chain variable region sequenceset forth as SEQ ID NO:4 and variable framework residues H24, H27, H29and H71 (Kabat numbering convention) from the monoclonal antibody 12B4heavy chain variable region sequence set forth as SEQ ID NO:4, whereinthe remainder of the heavy chain is from a human immunoglobulin, and(iii) a heavy chain comprising three complementarity determining regions(CDRs) from the monoclonal antibody 12B4 heavy chain variable regionsequence set forth as SEQ ID NO:4 and variable framework residues H24,H27, H29, H48, H71 and H78 (Kabat numbering convention) from themonoclonal antibody 12B4 heavy chain variable region sequence set forthas SEQ ID NO:4, wherein the remainder of the heavy chain is from a humanimmunoglobulin.
 14. The method of any one of claims 1, 4, 6 and 10,wherein the humanized immunoglobulin comprises a heavy and a light chainselected from the group consisting of: (a) a heavy chain variable regionsequence as set forth in residues 1-123 of SEQ ID NO:8 and a light chainvariable region sequence as set forth in residues 1-112 of SEQ ID NO:6;(b) a heavy chain variable region sequence as set forth in residues1-123 of SEQ ID NO:10, and a light chain variable region sequence as setforth in residues 1-112 of SEQ ID NO:6; and (c) a heavy chain variableregion sequence as set forth in residues 1-123 of SEQ ID NO:12 and alight chain variable region sequence as set forth in residues 1-112 ofSEQ ID NO:6.
 15. The method of any one of claims 1, 4, 6 and 10, whereinthe humanized immunoglobulin or an antigen binding fragment thereofbinds to beta amyloid peptide (Aβ) with a binding affinity of at least10⁷ M⁻¹.